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Thermo Fisher rnase a
RNase A Mass Mapping of the dsRNA using LC ESI MS. (a) Base peak chromatogram of the oligoribonucleotides generated from an RNase A digest of the purified dsRNA. Analysis was performed using LC ESI MS on a maXis ultra-high resolution time of flight instrument. A number of the identified oligoribonucleotides are highlighted. (b) Summary of the RNase mass mapping. Underlined bold = monoisotopic masses correspond to a number of theoretical sequence isomers in either sense or antisense strand. Bold = monoisotopic masses corresponding to a number of theoretical sequence isomers that are unique in either sense or antisense strand. Grey highlight = monoisotopic masses corresponding to single predicted unique oligoribonucleotide sequence in only the sense or antisense strand.
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1) Product Images from "Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry"

Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

Journal: Journal of Chromatography. a

doi: 10.1016/j.chroma.2016.12.062

RNase A Mass Mapping of the dsRNA using LC ESI MS. (a) Base peak chromatogram of the oligoribonucleotides generated from an RNase A digest of the purified dsRNA. Analysis was performed using LC ESI MS on a maXis ultra-high resolution time of flight instrument. A number of the identified oligoribonucleotides are highlighted. (b) Summary of the RNase mass mapping. Underlined bold = monoisotopic masses correspond to a number of theoretical sequence isomers in either sense or antisense strand. Bold = monoisotopic masses corresponding to a number of theoretical sequence isomers that are unique in either sense or antisense strand. Grey highlight = monoisotopic masses corresponding to single predicted unique oligoribonucleotide sequence in only the sense or antisense strand.
Figure Legend Snippet: RNase A Mass Mapping of the dsRNA using LC ESI MS. (a) Base peak chromatogram of the oligoribonucleotides generated from an RNase A digest of the purified dsRNA. Analysis was performed using LC ESI MS on a maXis ultra-high resolution time of flight instrument. A number of the identified oligoribonucleotides are highlighted. (b) Summary of the RNase mass mapping. Underlined bold = monoisotopic masses correspond to a number of theoretical sequence isomers in either sense or antisense strand. Bold = monoisotopic masses corresponding to a number of theoretical sequence isomers that are unique in either sense or antisense strand. Grey highlight = monoisotopic masses corresponding to single predicted unique oligoribonucleotide sequence in only the sense or antisense strand.

Techniques Used: Liquid Chromatography, Mass Spectrometry, Generated, Purification, Sequencing

RNase T1 Mass Mapping of the dsRNA using LC ESI MS. (a) Base peak chromatogram of the oligoribonucleotides generated from an RNase T1 digest of the purified dsRNA. Analysis was performed using LC ESI MS on a maXis ultra-high resolution time of flight instrument. A number of the identified oligoribonucleotides are highlighted. (b) Summary of the combined RNase A/T1 mass mapping. RNase A fragments are shown in red lines and RNase T1 in blue. Sequences highlighted in green = monoisotopic masses corresponding to a number of theoretical sequence isomers in either sense or antisense strand. Blue = monoisotopic masses corresponding to single predicted unique oligoribonucleotide sequence in only the sense or antisense strand from the RNase T1 digest. Red = monoisotopic masses corresponding to single predicted unique oligoribonucleotide sequence in only the sense or antisense strand from the RNase A digest. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: RNase T1 Mass Mapping of the dsRNA using LC ESI MS. (a) Base peak chromatogram of the oligoribonucleotides generated from an RNase T1 digest of the purified dsRNA. Analysis was performed using LC ESI MS on a maXis ultra-high resolution time of flight instrument. A number of the identified oligoribonucleotides are highlighted. (b) Summary of the combined RNase A/T1 mass mapping. RNase A fragments are shown in red lines and RNase T1 in blue. Sequences highlighted in green = monoisotopic masses corresponding to a number of theoretical sequence isomers in either sense or antisense strand. Blue = monoisotopic masses corresponding to single predicted unique oligoribonucleotide sequence in only the sense or antisense strand from the RNase T1 digest. Red = monoisotopic masses corresponding to single predicted unique oligoribonucleotide sequence in only the sense or antisense strand from the RNase A digest. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Liquid Chromatography, Mass Spectrometry, Generated, Purification, Sequencing

2) Product Images from "Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles"

Article Title: Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

Journal: Journal of Virology

doi: 10.1128/JVI.02921-15

SINV defective genomes interfere with full-length virus replication. (A) The number of full-length genomes in high-MOI passages of the SINV-WT (solid line) and SINV-G (dashed line) was assessed by qRT-PCR. Means and standard errors of the means are shown ( n  = 3). (B) 293T cells were transfected with a fixed amount of full-length SINV-WT (solid line) or SINV-G (dashed line) RNA and an increasing amount of Dg RNA. Titer was measured by plaque assay at 24 h posttransfection. Means and standard errors of the means are shown ( n  = 3). (C) Chikungunya virus replication-defective RNA (CHIK RNA) does not significantly interfere with SINV-WT or SINV-G virus replication compared to replication in infected cells with no additional RNA added (No RNA). Means and standard errors of the means are shown ( n  = 3; no significant differences were detected by a two-tailed student  t  test). (D) Viral supernatant from the transfection presented in panel B was subjected to RNase A treatment before qRT-PCR to measure the Dg/full-length genome ratio as previously described. The  x  axis indicates the ratio of Dg/full-length RNA initially transfected. Means and standard errors of the means are shown ( n  = 3). (E) 293T cells were transfected with a fixed amount of full-length SINV-WT (solid line) or SINV-G (dashed line) RNA and an increasing amount of Dg RNA mutated in its nsP1 starting codon (DgORF − ). Titer was measured by plaque assay at 24 h posttransfection. Means and standard errors of the means are shown ( n  = 3). (F and G) Stable cell lines expressing Dg, DgORF − , or CHIK3KB were infected with either SINV-WT (F) or SINV-G (G), and titers were measured at 16 h postinfection. Means and standard errors of the means are shown ( n  = 3; *,  P
Figure Legend Snippet: SINV defective genomes interfere with full-length virus replication. (A) The number of full-length genomes in high-MOI passages of the SINV-WT (solid line) and SINV-G (dashed line) was assessed by qRT-PCR. Means and standard errors of the means are shown ( n = 3). (B) 293T cells were transfected with a fixed amount of full-length SINV-WT (solid line) or SINV-G (dashed line) RNA and an increasing amount of Dg RNA. Titer was measured by plaque assay at 24 h posttransfection. Means and standard errors of the means are shown ( n = 3). (C) Chikungunya virus replication-defective RNA (CHIK RNA) does not significantly interfere with SINV-WT or SINV-G virus replication compared to replication in infected cells with no additional RNA added (No RNA). Means and standard errors of the means are shown ( n = 3; no significant differences were detected by a two-tailed student t test). (D) Viral supernatant from the transfection presented in panel B was subjected to RNase A treatment before qRT-PCR to measure the Dg/full-length genome ratio as previously described. The x axis indicates the ratio of Dg/full-length RNA initially transfected. Means and standard errors of the means are shown ( n = 3). (E) 293T cells were transfected with a fixed amount of full-length SINV-WT (solid line) or SINV-G (dashed line) RNA and an increasing amount of Dg RNA mutated in its nsP1 starting codon (DgORF − ). Titer was measured by plaque assay at 24 h posttransfection. Means and standard errors of the means are shown ( n = 3). (F and G) Stable cell lines expressing Dg, DgORF − , or CHIK3KB were infected with either SINV-WT (F) or SINV-G (G), and titers were measured at 16 h postinfection. Means and standard errors of the means are shown ( n = 3; *, P

Techniques Used: Quantitative RT-PCR, Transfection, Plaque Assay, Infection, Two Tailed Test, Stable Transfection, Expressing

Defectives particles can be detected during SINV-G infection. (A) Detection of SINV-WT (WT) and SINV-G (G) genomic sequences by RT-PCR after 24 h of infection at an MOI of 1 or 25.  In vitro -transcribed RNA (lane 1) was used as a control. The top band corresponds to the full-length genome. An extra low-molecular-weight band is indicated by an asterisk. (B) Viral supernatants from a 24-h infection with SINV-WT (lanes 4 and 5) or SINV-G (lanes 6 and 7) were either untreated or treated with RNase A (−, absence; +, presence) and then subjected to RT-PCR as described for panel A.  In vitro -transcribed RNA (lane 1) was used as an RT-PCR control. IVT RNAs incubated in solution (lane 2) and treated with RNase A (lane 3) were used as RNase controls. (C) Schematic of SINV full-length genome and the defective genome, Dg. Schematic shows the 5′ and 3′ untranslated regions with cap (C) and poly(A) tail and the intergenic region containing the subgenomic promoter (arrow) for structural proteins. Each protein-coding gene is depicted as a separate box, with positions of start and stop nucleotides numbered below. The deleted nucleotides are numbered below the Dg scheme. (D) Five undiluted passages were performed on BHK-21 cells with SINV-WT (lane 4) or SINV-G (lane 5) virus, or cells were mock infected (lane 3). Progeny virions were treated with RNase and purified by ultracentrifugation. Viral RNA was probed by Northern blotting using a probe specifically targeting the Dg breakpoint.  In vitro -transcribed (IVT) RNAs corresponding to full-length (FL) and Dg genomes, not treated with RNase, were used as positive controls for detection (lanes 1 and 2).
Figure Legend Snippet: Defectives particles can be detected during SINV-G infection. (A) Detection of SINV-WT (WT) and SINV-G (G) genomic sequences by RT-PCR after 24 h of infection at an MOI of 1 or 25. In vitro -transcribed RNA (lane 1) was used as a control. The top band corresponds to the full-length genome. An extra low-molecular-weight band is indicated by an asterisk. (B) Viral supernatants from a 24-h infection with SINV-WT (lanes 4 and 5) or SINV-G (lanes 6 and 7) were either untreated or treated with RNase A (−, absence; +, presence) and then subjected to RT-PCR as described for panel A. In vitro -transcribed RNA (lane 1) was used as an RT-PCR control. IVT RNAs incubated in solution (lane 2) and treated with RNase A (lane 3) were used as RNase controls. (C) Schematic of SINV full-length genome and the defective genome, Dg. Schematic shows the 5′ and 3′ untranslated regions with cap (C) and poly(A) tail and the intergenic region containing the subgenomic promoter (arrow) for structural proteins. Each protein-coding gene is depicted as a separate box, with positions of start and stop nucleotides numbered below. The deleted nucleotides are numbered below the Dg scheme. (D) Five undiluted passages were performed on BHK-21 cells with SINV-WT (lane 4) or SINV-G (lane 5) virus, or cells were mock infected (lane 3). Progeny virions were treated with RNase and purified by ultracentrifugation. Viral RNA was probed by Northern blotting using a probe specifically targeting the Dg breakpoint. In vitro -transcribed (IVT) RNAs corresponding to full-length (FL) and Dg genomes, not treated with RNase, were used as positive controls for detection (lanes 1 and 2).

Techniques Used: Infection, Genomic Sequencing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Molecular Weight, Incubation, Purification, Northern Blot

3) Product Images from "RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells"

Article Title: RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0129806

IL-12 production from human (A) monocytes and (B) monocyte-depleted PBMCs against  E .  faecalis  EC-12. Heat-killed  E .  faecalis  EC-12 was treated with or without RNase A in high salt buffer and co-cultured with  (A)  monocytes or  (B)  monocyte-depleted PBMCs for 24 h. The IL-12 protein concentration in the culture supernatant was measured by enzyme-linked immunosorbent assay. Bars sharing the same letter are not significantly different at P
Figure Legend Snippet: IL-12 production from human (A) monocytes and (B) monocyte-depleted PBMCs against E . faecalis EC-12. Heat-killed E . faecalis EC-12 was treated with or without RNase A in high salt buffer and co-cultured with (A) monocytes or (B) monocyte-depleted PBMCs for 24 h. The IL-12 protein concentration in the culture supernatant was measured by enzyme-linked immunosorbent assay. Bars sharing the same letter are not significantly different at P

Techniques Used: Cell Culture, Protein Concentration, Enzyme-linked Immunosorbent Assay

Effect of nuclease treatment of LAB on IL-12-inducing ability in human PBMCs. Heat-killed  L .  gasseri  JCM5344,  E .  faecalis  EC-12, and  B .  breve  JCM1192 were treated with or without nuclease and co-cultured with PBMCs obtained from three donors, for 24 h. The IL-12 protein concentration in the culture supernatant was measured by enzyme-linked immunosorbent assay. Data are expressed as relative percentage to non-treated LAB. The value above the bars of non-treated LAB indicates the mean value of IL-12 concentration (pg/mL) in each donor.  n : non-treated,  RNase A (L):  RNase A treatment in low salt buffer (digests all RNA),  RNase A (H):  RNase A treatment in high salt buffer (digests ssRNA),  Cq : chloroquine treatment of PBMCs prior to bacterial stimulation. **: P
Figure Legend Snippet: Effect of nuclease treatment of LAB on IL-12-inducing ability in human PBMCs. Heat-killed L . gasseri JCM5344, E . faecalis EC-12, and B . breve JCM1192 were treated with or without nuclease and co-cultured with PBMCs obtained from three donors, for 24 h. The IL-12 protein concentration in the culture supernatant was measured by enzyme-linked immunosorbent assay. Data are expressed as relative percentage to non-treated LAB. The value above the bars of non-treated LAB indicates the mean value of IL-12 concentration (pg/mL) in each donor. n : non-treated, RNase A (L): RNase A treatment in low salt buffer (digests all RNA), RNase A (H): RNase A treatment in high salt buffer (digests ssRNA), Cq : chloroquine treatment of PBMCs prior to bacterial stimulation. **: P

Techniques Used: Cell Culture, Protein Concentration, Enzyme-linked Immunosorbent Assay, Concentration Assay

4) Product Images from "Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes"

Article Title: Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006197

PV infectivity is not affected by covalent linkage of RNase A to the virus. A)  RNase A is covalently linked to PV VP1. Conjugation reactions containing  35 S-Met/Cys radiolabelled PV and/or RNase A (as indicated at the top of the image) were subjected to SDS-PAGE and western blot with antisera against RNase A. The major over-exposed bands correspond to RNase A monomer and dimer (indicated by arrows). Bands in the middle lane are the expected size for radiolabelled virus proteins VP1, VP2 and VP3. The upper band in the left hand lane is the expected size for RNase A covalently attached to VP1 (as indicated by arrow). Molecular weight standards (kDa) are shown on the left.  B)  Plaque assay of PV conjugated to RNase A (0, 90, 300, 600 molar ratio). Plaque forming units (pfu) were expressed as percentage of no RNase A control and data pooled from three independent experiments.  C)  Ribonuclease activity was measured by quantifying tRNA fluorescence (Relative Fluorescence Units, RFU) with Ribogreen in the presence of RNase A, purified PV, PV conjugated to RNase A with EDC (PV-A + EDC) and mock conjugation reaction (PV-A—EDC).  D)  Sucrose gradient profile of  3 H-U-PV RNA (as in   Fig 3E ) uncoated  in vitro  at 50°C for 10 min in the presence of RNase A (1mg/ml) or PBS carrier control (representative of two independent experiments).  E)  Sucrose gradient profile (as in   Fig 3E ) of  3 H-U-PV RNA from viral particles directly conjugated to RNase A with the cross-linker EDC (PV-A + EDC) or mock conjugated (no EDC, PV-A—EDC) uncoated  in vitro  at 50°C for 10 min (representative of two independent experiments).  F)  Representative image of HeLa Ohio cells infected with PV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.92 +/- 0.06 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm.  G)  RNase activity (as in C) of individual and mixed components of the RNase S system.  H)  RNase activity (as in C and G) and  I)  virus titre (as in B) of PV conjugated to individual or mixed components of the RNase S system. (All data from three independent experiments with error bars showing standard error, unless stated).
Figure Legend Snippet: PV infectivity is not affected by covalent linkage of RNase A to the virus. A) RNase A is covalently linked to PV VP1. Conjugation reactions containing 35 S-Met/Cys radiolabelled PV and/or RNase A (as indicated at the top of the image) were subjected to SDS-PAGE and western blot with antisera against RNase A. The major over-exposed bands correspond to RNase A monomer and dimer (indicated by arrows). Bands in the middle lane are the expected size for radiolabelled virus proteins VP1, VP2 and VP3. The upper band in the left hand lane is the expected size for RNase A covalently attached to VP1 (as indicated by arrow). Molecular weight standards (kDa) are shown on the left. B) Plaque assay of PV conjugated to RNase A (0, 90, 300, 600 molar ratio). Plaque forming units (pfu) were expressed as percentage of no RNase A control and data pooled from three independent experiments. C) Ribonuclease activity was measured by quantifying tRNA fluorescence (Relative Fluorescence Units, RFU) with Ribogreen in the presence of RNase A, purified PV, PV conjugated to RNase A with EDC (PV-A + EDC) and mock conjugation reaction (PV-A—EDC). D) Sucrose gradient profile of 3 H-U-PV RNA (as in Fig 3E ) uncoated in vitro at 50°C for 10 min in the presence of RNase A (1mg/ml) or PBS carrier control (representative of two independent experiments). E) Sucrose gradient profile (as in Fig 3E ) of 3 H-U-PV RNA from viral particles directly conjugated to RNase A with the cross-linker EDC (PV-A + EDC) or mock conjugated (no EDC, PV-A—EDC) uncoated in vitro at 50°C for 10 min (representative of two independent experiments). F) Representative image of HeLa Ohio cells infected with PV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.92 +/- 0.06 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. G) RNase activity (as in C) of individual and mixed components of the RNase S system. H) RNase activity (as in C and G) and I) virus titre (as in B) of PV conjugated to individual or mixed components of the RNase S system. (All data from three independent experiments with error bars showing standard error, unless stated).

Techniques Used: Infection, Conjugation Assay, SDS Page, Western Blot, Molecular Weight, Plaque Assay, Activity Assay, Fluorescence, Purification, In Vitro, Staining

ERAV is co-internalized with RNase A but infectivity is not compromised. A)  Representative images of HeLa Ohio cells infected with ERAV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.86 +/- 0.09 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm.  B)  Plaque assay of ERAV in the presence of 0–1 mg/ml RNase A. Plaque forming units (pfu) were expressed as percentage of no RNase A control. Data are from three independent experiments with error bars showing standard error.
Figure Legend Snippet: ERAV is co-internalized with RNase A but infectivity is not compromised. A) Representative images of HeLa Ohio cells infected with ERAV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.86 +/- 0.09 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. B) Plaque assay of ERAV in the presence of 0–1 mg/ml RNase A. Plaque forming units (pfu) were expressed as percentage of no RNase A control. Data are from three independent experiments with error bars showing standard error.

Techniques Used: Infection, Staining, Plaque Assay

Receptor-decorated liposomes containing fluorescent dye detect PV RNA release. A)  Representative images of YoPro-1 encapsulating receptor-decorated liposomes (YRDLs) complexed with PV in the presence or absence of RNase A (50 μg/ml). Note that RNase A was added to the extra-liposomal space after PV-YRDL complexes were formed, but prior to heating the samples for 20 min at 37°C. Images were collected at room temperature using a 20X objective as described in Materials and Methods. Scale bars are 200 μm.  B)  Normalized histograms showing the number of pixels (y-axis) with a given level of fluorescence (in arbitrary units) (x-axis) of PV-YRDL complexes shown in A in the absence (green curve) and presence (black curve) of RNase A. ( C and D)  Representative images of PV RNA (in the absence of liposomes) in the presence of YoPro-1 dye following induction of uncoating by sPVR at 37°C (C) or by heating at 52°C (D) for 20 min in the presence or absence of RNase A (50 μg/ml). Images were collected using a 100X objective as described in Materials and Methods. Scale bars are 40 μm.  E)  Representative still frames from a time lapse of PV-YRDLs gradually heated from room temperature to 42°C. Average time lapse for averaged image is indicated. After 15 min of imaging a single field of view, a second region of interest was imaged in order to evaluate the influence of photobleaching on the fluorescence intensity (second ROI at 20 min shown on the right-side panel). Images were captured at 100x magnification using a custom built Total Internal Reflectance Fluorescence Microscopy (TIR-FM) setup, attached to an Olympus IX-71 microscope, as described in Material and Methods. Scale bars are 5 μm.  F)  PV-YRDLs integrated fluorescence intensity obtained as indicated in Materials and Methods (expressed as fold change of T = 1 min, left y-axis) during a 20 min time course (time in min along the x-axis) when the sample was heated from room temperature to 42°C. The temperature of the lens (right y-axis) is shown as a function of time (grey dashed line). Because the objective lens and the sample are 1.18 mm apart (with oil connecting the lens to the sample slide), the temperature of the lens is used to estimate the temperature of the sample. 42°C is the upper limit of the imaging apparatus. The black triangle shows the integrated fluorescence intensity of a region of interest that was imaged at a single time point of 20 min in order to assess photobleaching.  G)  Representative images of YoPro-1 encapsulating RDL using the same microscope setup described for E. YRDLs were incubated at 37°C for 10 min, alone with no PV (left), or were pre-incubated with PV at room temperature for 10 min to allow complex formation, and then incubated 37°C for 10 min (right). Scale bars are 5 μm.
Figure Legend Snippet: Receptor-decorated liposomes containing fluorescent dye detect PV RNA release. A) Representative images of YoPro-1 encapsulating receptor-decorated liposomes (YRDLs) complexed with PV in the presence or absence of RNase A (50 μg/ml). Note that RNase A was added to the extra-liposomal space after PV-YRDL complexes were formed, but prior to heating the samples for 20 min at 37°C. Images were collected at room temperature using a 20X objective as described in Materials and Methods. Scale bars are 200 μm. B) Normalized histograms showing the number of pixels (y-axis) with a given level of fluorescence (in arbitrary units) (x-axis) of PV-YRDL complexes shown in A in the absence (green curve) and presence (black curve) of RNase A. ( C and D) Representative images of PV RNA (in the absence of liposomes) in the presence of YoPro-1 dye following induction of uncoating by sPVR at 37°C (C) or by heating at 52°C (D) for 20 min in the presence or absence of RNase A (50 μg/ml). Images were collected using a 100X objective as described in Materials and Methods. Scale bars are 40 μm. E) Representative still frames from a time lapse of PV-YRDLs gradually heated from room temperature to 42°C. Average time lapse for averaged image is indicated. After 15 min of imaging a single field of view, a second region of interest was imaged in order to evaluate the influence of photobleaching on the fluorescence intensity (second ROI at 20 min shown on the right-side panel). Images were captured at 100x magnification using a custom built Total Internal Reflectance Fluorescence Microscopy (TIR-FM) setup, attached to an Olympus IX-71 microscope, as described in Material and Methods. Scale bars are 5 μm. F) PV-YRDLs integrated fluorescence intensity obtained as indicated in Materials and Methods (expressed as fold change of T = 1 min, left y-axis) during a 20 min time course (time in min along the x-axis) when the sample was heated from room temperature to 42°C. The temperature of the lens (right y-axis) is shown as a function of time (grey dashed line). Because the objective lens and the sample are 1.18 mm apart (with oil connecting the lens to the sample slide), the temperature of the lens is used to estimate the temperature of the sample. 42°C is the upper limit of the imaging apparatus. The black triangle shows the integrated fluorescence intensity of a region of interest that was imaged at a single time point of 20 min in order to assess photobleaching. G) Representative images of YoPro-1 encapsulating RDL using the same microscope setup described for E. YRDLs were incubated at 37°C for 10 min, alone with no PV (left), or were pre-incubated with PV at room temperature for 10 min to allow complex formation, and then incubated 37°C for 10 min (right). Scale bars are 5 μm.

Techniques Used: Fluorescence, Imaging, Microscopy, Incubation

PV infectivity and RNA integrity are not affected by the presence of high levels of RNase A during the infection process. A)  Representative image of HeLa Ohio cells infected with PV-Cy2 (green) in the presence of Dextrans-10 kDa conjugated to Alexa-594 (red) fixed 15 min post-infection. The degree of co-internalization (right-hand side panel) was measured on 10 random cells, R = 0.89 +/- 0.09 (SD). Scale bar is 5 μm.  B)  Plaque assay of PV in the presence of 0–1 mg/ml RNase A. Plaque forming units were expressed as percentage of no RNase A control.  C)  Scintillation counting of internalized vs unattached  3 H-U-PV in HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control.  D)  Scintillation counting of recovered and flow-through samples after a column-based RNA purification procedure of  3 H-U-PV RNA internalized into HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control.  E)  Scintillation counting of sucrose density gradient (15–30% sucrose, 0.1% SDS, 0.1 M Na acetate. Fraction 1 = top, 15% sucrose) of  3 H-U-PV RNA recovered from HeLa Ohio cells 30 min post-infection in the presence or absence of 1 mg/ml RNase A (PV+HeLa+A, red line, and PV+HeLa, blue line, respectively). Data is expressed as percentage of the total counts per minutes (cpm) loaded onto the gradient. All data are from three independent experiments and error bars show standard error.
Figure Legend Snippet: PV infectivity and RNA integrity are not affected by the presence of high levels of RNase A during the infection process. A) Representative image of HeLa Ohio cells infected with PV-Cy2 (green) in the presence of Dextrans-10 kDa conjugated to Alexa-594 (red) fixed 15 min post-infection. The degree of co-internalization (right-hand side panel) was measured on 10 random cells, R = 0.89 +/- 0.09 (SD). Scale bar is 5 μm. B) Plaque assay of PV in the presence of 0–1 mg/ml RNase A. Plaque forming units were expressed as percentage of no RNase A control. C) Scintillation counting of internalized vs unattached 3 H-U-PV in HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. D) Scintillation counting of recovered and flow-through samples after a column-based RNA purification procedure of 3 H-U-PV RNA internalized into HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. E) Scintillation counting of sucrose density gradient (15–30% sucrose, 0.1% SDS, 0.1 M Na acetate. Fraction 1 = top, 15% sucrose) of 3 H-U-PV RNA recovered from HeLa Ohio cells 30 min post-infection in the presence or absence of 1 mg/ml RNase A (PV+HeLa+A, red line, and PV+HeLa, blue line, respectively). Data is expressed as percentage of the total counts per minutes (cpm) loaded onto the gradient. All data are from three independent experiments and error bars show standard error.

Techniques Used: Infection, Plaque Assay, Flow Cytometry, Purification

5) Product Images from "RNase H1 directs origin-specific initiation of DNA replication in human mitochondria"

Article Title: RNase H1 directs origin-specific initiation of DNA replication in human mitochondria

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1007781

Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.
Figure Legend Snippet: Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.

Techniques Used: In Vitro, Purification, Recombinant, Staining, SDS Page, Labeling, Plasmid Preparation, Sequencing, Concentration Assay

6) Product Images from "Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts"

Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014411

Analysis of the radiolabelled RNA sensitivity to RNase A. RNA polymerization reactions were performed with axolotl LSE and [α– 32 P] CTP in the presence of exogenous sense 534 bases PKCζ synthetic transcript (A); at the indicated incubation times, total RNA was extracted, solubilized in water then either directly mixed with the loading buffer (lanes 1, 2) or diluted in the hybridizing buffer (80% formamide, lanes 3–18). Sense 534 bases PKCζ synthetic transcript was incubated in control hybridization reactions (B), either alone (lanes 9–13) or in the presence of a 540 bases synthetic RNA (antisense) complementary to the sense 534 bases transcript (lanes 14–18). RNAs were heat-denaturated then incubated overnight at 65°C. Following the denaturation/hybridization steps, the ability of the RNAs to resist to RNase A catalyzed hydrolysis was studied in the digestion buffer (8% formamide) and RNase A at final concentrations of 1 ng/mL (lanes 5, 10, 15), 10 ng/mL (lanes 6, 11, 16), 100 ng/mL (lanes 7, 12, 17), 1 µg/mL (lanes 8, 13, 18) or in the absence of RNase A (lanes 3, 4, 9, 14). RNAs were analyzed in polyacrylamide 8 M-urea gels that were stained with ≪Stains-all≫ and exposed to a PhosphoImager screen when mentioned. As compared to the RNase A ≪-≫ conditions (lane 4), the percent of residual radioactivity observed at the migration length of the sense 534 bases transcript (arrow head) has been quantitated : lane 5, 99%; lane 6, 40%; lane 7, 3%; lane 8, 1%. The sizes of the RNA molecular Markers Low Range ladder are indicated in bases (b).
Figure Legend Snippet: Analysis of the radiolabelled RNA sensitivity to RNase A. RNA polymerization reactions were performed with axolotl LSE and [α– 32 P] CTP in the presence of exogenous sense 534 bases PKCζ synthetic transcript (A); at the indicated incubation times, total RNA was extracted, solubilized in water then either directly mixed with the loading buffer (lanes 1, 2) or diluted in the hybridizing buffer (80% formamide, lanes 3–18). Sense 534 bases PKCζ synthetic transcript was incubated in control hybridization reactions (B), either alone (lanes 9–13) or in the presence of a 540 bases synthetic RNA (antisense) complementary to the sense 534 bases transcript (lanes 14–18). RNAs were heat-denaturated then incubated overnight at 65°C. Following the denaturation/hybridization steps, the ability of the RNAs to resist to RNase A catalyzed hydrolysis was studied in the digestion buffer (8% formamide) and RNase A at final concentrations of 1 ng/mL (lanes 5, 10, 15), 10 ng/mL (lanes 6, 11, 16), 100 ng/mL (lanes 7, 12, 17), 1 µg/mL (lanes 8, 13, 18) or in the absence of RNase A (lanes 3, 4, 9, 14). RNAs were analyzed in polyacrylamide 8 M-urea gels that were stained with ≪Stains-all≫ and exposed to a PhosphoImager screen when mentioned. As compared to the RNase A ≪-≫ conditions (lane 4), the percent of residual radioactivity observed at the migration length of the sense 534 bases transcript (arrow head) has been quantitated : lane 5, 99%; lane 6, 40%; lane 7, 3%; lane 8, 1%. The sizes of the RNA molecular Markers Low Range ladder are indicated in bases (b).

Techniques Used: Incubation, Hybridization, Staining, Radioactivity, Migration

Discrete radiolabelled RNA bands are detected after incubation of the axolotl and  Xenopus  extracts in the presence of [α– 32 P] CTP or [α– 32 P] UTP. Polymerization reactions were performed using axolotl or  Xenopus  LSE with either [α– 32 P] CTP or [α– 32 P] UTP (lane 4) and in the presence (lane 6) or not of 200 µg/mL α-amanitine, an RNA pol II and pol III inhibitor. After the indicated incubation time, samples were treated at 37°C during 1 hr with RNase A (lane 5) or during 15 min with RQ1 DNase (lane 8) or not (lanes 1–4, 6, 7, 9–11). RNA was extracted, electrophorezed through a denaturating agarose gel and visualized after ethidium bromide staining and UV illumination; the gel was dried and autoradiographied. Migration lengths of RNA size markers are indicated in bases (b).
Figure Legend Snippet: Discrete radiolabelled RNA bands are detected after incubation of the axolotl and Xenopus extracts in the presence of [α– 32 P] CTP or [α– 32 P] UTP. Polymerization reactions were performed using axolotl or Xenopus LSE with either [α– 32 P] CTP or [α– 32 P] UTP (lane 4) and in the presence (lane 6) or not of 200 µg/mL α-amanitine, an RNA pol II and pol III inhibitor. After the indicated incubation time, samples were treated at 37°C during 1 hr with RNase A (lane 5) or during 15 min with RQ1 DNase (lane 8) or not (lanes 1–4, 6, 7, 9–11). RNA was extracted, electrophorezed through a denaturating agarose gel and visualized after ethidium bromide staining and UV illumination; the gel was dried and autoradiographied. Migration lengths of RNA size markers are indicated in bases (b).

Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, Migration

A radiolabelled RNA comigrates with the exogenous RNA added to the extract. Reactions were performed during 4 hr with axolotl (A : lanes 1–3. C : lanes 8–13) or  Xenopus  (B : lanes 4–7) LSE in the presence of [α– 32 P] CTP, in the presence (+) or absence (−) of 10 µg of  in vitro  transcribed  Xenopus  globin 3′UTR poly (A) −  (555 b; lanes 1–3 and 8–13) or poly (A) +  (620 b; lanes 4–7) RNA and in the presence of 2.5 mM cordycepin (lane 12) or cordycepin 5′-triphosphate (lane 13) or not (lanes 1–11). After incubation, samples were treated at 37°C during 1 hr with 10 mg/mL RNase A (lane 10) or not, RNA was extracted and processed according to the same protocol as in   figure 3 . The radioactivity corresponding to 1/10 of the reaction analyzed by TCA precipitation is indicated in A (counts per minute : cpm).  In vitro  synthesized  Xenopus  globin 3′UTR poly(A) −  (555 b) or poly(A) +  (620 b) RNA analyzed in parallel are shown in the UV part of A and C respectively. Migration lengths of RNA size markers (M lanes in C) are indicated in bases (b).
Figure Legend Snippet: A radiolabelled RNA comigrates with the exogenous RNA added to the extract. Reactions were performed during 4 hr with axolotl (A : lanes 1–3. C : lanes 8–13) or Xenopus (B : lanes 4–7) LSE in the presence of [α– 32 P] CTP, in the presence (+) or absence (−) of 10 µg of in vitro transcribed Xenopus globin 3′UTR poly (A) − (555 b; lanes 1–3 and 8–13) or poly (A) + (620 b; lanes 4–7) RNA and in the presence of 2.5 mM cordycepin (lane 12) or cordycepin 5′-triphosphate (lane 13) or not (lanes 1–11). After incubation, samples were treated at 37°C during 1 hr with 10 mg/mL RNase A (lane 10) or not, RNA was extracted and processed according to the same protocol as in figure 3 . The radioactivity corresponding to 1/10 of the reaction analyzed by TCA precipitation is indicated in A (counts per minute : cpm). In vitro synthesized Xenopus globin 3′UTR poly(A) − (555 b) or poly(A) + (620 b) RNA analyzed in parallel are shown in the UV part of A and C respectively. Migration lengths of RNA size markers (M lanes in C) are indicated in bases (b).

Techniques Used: In Vitro, Incubation, Radioactivity, TCA Precipitation, Synthesized, Migration

7) Product Images from "Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication"

Article Title: Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

Journal:

doi: 10.1128/JVI.01647-08

Enzymatic and chemical probing of the 5′-terminal stem-loop of the DENV genome. (A) An RNA corresponding to the first 160 nucleotides of the viral genome was subjected to RNase A, RNase PhyM, RNase V 1 , or NMIA treatment, and the cleaved or modified
Figure Legend Snippet: Enzymatic and chemical probing of the 5′-terminal stem-loop of the DENV genome. (A) An RNA corresponding to the first 160 nucleotides of the viral genome was subjected to RNase A, RNase PhyM, RNase V 1 , or NMIA treatment, and the cleaved or modified

Techniques Used: Modification

Functional in vivo and in vitro compatibility of SLAs from different DENV serotypes. (A) Enzymatic probing of the first 70 nucleotides of DENV1 and DENV2. The RNAs were subjected to RNase V 1 (V) or RNase A (A) digestion, and the cleaved RNAs were analyzed
Figure Legend Snippet: Functional in vivo and in vitro compatibility of SLAs from different DENV serotypes. (A) Enzymatic probing of the first 70 nucleotides of DENV1 and DENV2. The RNAs were subjected to RNase V 1 (V) or RNase A (A) digestion, and the cleaved RNAs were analyzed

Techniques Used: Functional Assay, In Vivo, In Vitro

8) Product Images from "Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA"

Article Title: Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx051

Monomethylation of 5΄-monophosphate of tRNA His  by BCDIN3D  in vitro . ( A )  In vitro  methylation of tRNA His  and pre-miR145 transcripts. Time courses of methyl-group transfer from AdoMet to tRNA His  and pre-miR145 transcripts under standard conditions (2 μM RNA substrate and 0.1 μM recombinant BCDIN3D). ( B )  In vitro  methylation of tRNA His  and pre-miR145 with a higher concentration of BCDIN3D (1 μM) at 37°C for 2 h. After the reaction, the RNA was separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue (left), dried and exposed to a BASS2000 imaging plate (Fujifilm, Japan) for 12 hours (right). Mass spectrometric analysis of the RNase A-digested products of BCDIN3D-treated. ( C ) tRNA His  and ( D ) pre-miR145. ( E ) The steady state kinetics of methylation of tRNA His  and pre-miR145. Reaction mixtures containing various concentrations of tRNA His  (0.125–4 μM; left) and pre-miR145 (1–15 μM; right) were incubated at 37°C for 10 min. The bars in the graphs are SD of more than two independent experiments.
Figure Legend Snippet: Monomethylation of 5΄-monophosphate of tRNA His by BCDIN3D in vitro . ( A ) In vitro methylation of tRNA His and pre-miR145 transcripts. Time courses of methyl-group transfer from AdoMet to tRNA His and pre-miR145 transcripts under standard conditions (2 μM RNA substrate and 0.1 μM recombinant BCDIN3D). ( B ) In vitro methylation of tRNA His and pre-miR145 with a higher concentration of BCDIN3D (1 μM) at 37°C for 2 h. After the reaction, the RNA was separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue (left), dried and exposed to a BASS2000 imaging plate (Fujifilm, Japan) for 12 hours (right). Mass spectrometric analysis of the RNase A-digested products of BCDIN3D-treated. ( C ) tRNA His and ( D ) pre-miR145. ( E ) The steady state kinetics of methylation of tRNA His and pre-miR145. Reaction mixtures containing various concentrations of tRNA His (0.125–4 μM; left) and pre-miR145 (1–15 μM; right) were incubated at 37°C for 10 min. The bars in the graphs are SD of more than two independent experiments.

Techniques Used: In Vitro, Methylation, Recombinant, Concentration Assay, Polyacrylamide Gel Electrophoresis, Staining, Imaging, Incubation

Cytoplasmic tRNA His  binds to BCDIN3D or its associated protein(s) and has 5΄-monomethylmonophosphate. ( A ) Schematic presentation of the isolation of RNAs interacting with BCDIN3D. ( B ) Electrophoretic analysis of the RNA fraction co-purified with SBP-BCDIN3D (SBP-BCDIN3D, right lane), and control SBP (SBP, left lane) from cell extracts. The arrow indicates RNA-x specifically bound to BCDIN3D or its associated protein(s). ( C ) RT-PCR of RNAs interacting with BCDIN3D. Total RNAs from SBP-BCDIN3D- or SBP-expressing cells or RNAs co-purified with SBP-BCDIN3D or SBP were subjected to RT-PCR, using tRNA His - (left) or tRNA Phe - (right) specific primers. ( D ) Nucleotide sequence of human cytoplasmic tRNA His  (  23 ). LC/MS analysis of RNase T1-digested fragments of RNA-x identified cytoplasmic tRNA His  (  Supplementary Figure S1A–C ). The fragments cover the sequences of cytoplasmic tRNA His  (grey-shaded). ( E ) LC/MS analysis of RNase A-digested fragments of RNA-x. Identification of the molecular mass corresponding to 5΄pmG -1 -G 1 -C 2  p (pmG: guanosine 5΄-monomethyl monophosphate;  m/z  1,106) of cytoplasmic tRNA His  (  Supplementary Figure S1D ). ( F ) Collision-induced dissociation (CID) spectrum of the RNA fragment of 5΄pmG -1 -G 1 -C 2  p in (E), showing that a methyl-group is attached to the 5΄-monophosphate of tRNA His .
Figure Legend Snippet: Cytoplasmic tRNA His binds to BCDIN3D or its associated protein(s) and has 5΄-monomethylmonophosphate. ( A ) Schematic presentation of the isolation of RNAs interacting with BCDIN3D. ( B ) Electrophoretic analysis of the RNA fraction co-purified with SBP-BCDIN3D (SBP-BCDIN3D, right lane), and control SBP (SBP, left lane) from cell extracts. The arrow indicates RNA-x specifically bound to BCDIN3D or its associated protein(s). ( C ) RT-PCR of RNAs interacting with BCDIN3D. Total RNAs from SBP-BCDIN3D- or SBP-expressing cells or RNAs co-purified with SBP-BCDIN3D or SBP were subjected to RT-PCR, using tRNA His - (left) or tRNA Phe - (right) specific primers. ( D ) Nucleotide sequence of human cytoplasmic tRNA His ( 23 ). LC/MS analysis of RNase T1-digested fragments of RNA-x identified cytoplasmic tRNA His ( Supplementary Figure S1A–C ). The fragments cover the sequences of cytoplasmic tRNA His (grey-shaded). ( E ) LC/MS analysis of RNase A-digested fragments of RNA-x. Identification of the molecular mass corresponding to 5΄pmG -1 -G 1 -C 2 p (pmG: guanosine 5΄-monomethyl monophosphate; m/z 1,106) of cytoplasmic tRNA His ( Supplementary Figure S1D ). ( F ) Collision-induced dissociation (CID) spectrum of the RNA fragment of 5΄pmG -1 -G 1 -C 2 p in (E), showing that a methyl-group is attached to the 5΄-monophosphate of tRNA His .

Techniques Used: Isolation, Purification, Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing, Liquid Chromatography, Mass Spectrometry

BCDIN3D monomethylates the 5΄-monophosphate of tRNA His in vivo . ( A ) Schematic representation of the location of guide RNAs (gRNA1 and gRNA2) targeting the BCDIN3D gene locus. The nucleotide sequence of the region surrounding the target site (upper), and those of the regions surrounding the targeted deletion sites in knockout cells (KO1 and KO2, lower) for cleavage by CRISPR/Cas9. ( B ) Expression of the BCDIN3D protein in wild-type HEK293T cells and BCDIN3D knockout cells (KO1 and KO2). The BCDIN3D protein was detected by western blotting, using an anti-BCDIN3D antibody. GAPDH expression was used as a positive control. ( C ) Small RNA fractions were prepared from wild-type HEK293T cells and BCDIN3D KO cells (KO1 and KO2). The RNAs were subjected to  in vitro  methylation by BCDIN3D, as in Figure   2B , using 5 μg small RNA fractions, and the reaction products were separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue and dried (left). The  14 C-labeled RNAs were detected with a phosphorimager (right). ( D )  In vitro  methylations of tRNA His  species isolated from HEK293T, KO1 and KO2 cells, and those of tRNA His  species from rescued KO1 and KO2 cells with exogenous expression of BCDIN3D (KO1+BCDIN3D and KO2+BCDIN3D). tRNA His  species after the reaction were separated by polyacrylamide gel electrophoresis, and the gel was stained with toluidine blue (upper). The  14 C-labeled tRNA His  species were detected with a phosphorimager (lower), and the relative  14 C-band intensities were calculated. The intensity of tRNA His  from KO1 was designated as 100. ( E, F ) LC/MS analysis of tRNA His  isolated from KO1 and KO2. The RNase A-digested fragments of tRNA His  were analyzed by LC/MS. The 5΄-mpG -1 G 1 C 2 p is not detected in tRNA His  from KO1 and KO2. The 5΄-mpG -1 G 1 C 2 p was partially restored by the exogenous expression of BCDIN3D in the KO cells (KO1+BCDIN3D and KO2+BCDIN3D).
Figure Legend Snippet: BCDIN3D monomethylates the 5΄-monophosphate of tRNA His in vivo . ( A ) Schematic representation of the location of guide RNAs (gRNA1 and gRNA2) targeting the BCDIN3D gene locus. The nucleotide sequence of the region surrounding the target site (upper), and those of the regions surrounding the targeted deletion sites in knockout cells (KO1 and KO2, lower) for cleavage by CRISPR/Cas9. ( B ) Expression of the BCDIN3D protein in wild-type HEK293T cells and BCDIN3D knockout cells (KO1 and KO2). The BCDIN3D protein was detected by western blotting, using an anti-BCDIN3D antibody. GAPDH expression was used as a positive control. ( C ) Small RNA fractions were prepared from wild-type HEK293T cells and BCDIN3D KO cells (KO1 and KO2). The RNAs were subjected to in vitro methylation by BCDIN3D, as in Figure 2B , using 5 μg small RNA fractions, and the reaction products were separated by 10% (v/v) polyacrylamide gel electrophoresis under denaturing conditions. The gel was stained with toluidine blue and dried (left). The 14 C-labeled RNAs were detected with a phosphorimager (right). ( D ) In vitro methylations of tRNA His species isolated from HEK293T, KO1 and KO2 cells, and those of tRNA His species from rescued KO1 and KO2 cells with exogenous expression of BCDIN3D (KO1+BCDIN3D and KO2+BCDIN3D). tRNA His species after the reaction were separated by polyacrylamide gel electrophoresis, and the gel was stained with toluidine blue (upper). The 14 C-labeled tRNA His species were detected with a phosphorimager (lower), and the relative 14 C-band intensities were calculated. The intensity of tRNA His from KO1 was designated as 100. ( E, F ) LC/MS analysis of tRNA His isolated from KO1 and KO2. The RNase A-digested fragments of tRNA His were analyzed by LC/MS. The 5΄-mpG -1 G 1 C 2 p is not detected in tRNA His from KO1 and KO2. The 5΄-mpG -1 G 1 C 2 p was partially restored by the exogenous expression of BCDIN3D in the KO cells (KO1+BCDIN3D and KO2+BCDIN3D).

Techniques Used: In Vivo, Sequencing, Knock-Out, CRISPR, Expressing, Western Blot, Positive Control, Gene Knockout, In Vitro, Methylation, Polyacrylamide Gel Electrophoresis, Staining, Labeling, Isolation, Liquid Chromatography, Mass Spectrometry

9) Product Images from "The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera"

Article Title: The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera

Journal: PLoS ONE

doi: 10.1371/journal.pone.0151035

Immunolocalization of HEX 110 in RNase A-treated ovarioles (stage S2) of queenright nurse workers. (A) Control ovariole non-incubated with RNase A. HEX 110 green foci (labeled with anti-HEX 110/Alexa-Fluor 488) are abundant in the nucleus (arrowheads) and cytoplasm (arrows) of nurse (Nc) and follicle (Fc) cells surrounding the oocyte (stage 2, see   Fig 2 ). (B) Ovariole incubated with RNase A shows scarce HEX 110 foci (arrows). DAPI-staining (blue) in B highlights the small nuclei of follicle cells (Fc) that surround the oocyte (stage 1, see   Fig 2 ) and are interspersed between the large nurse cell (Nc) nuclei. Quantification of pixels intensity in the selected areas (dashed rectangles) of the images obtained from RNase A-treated and untreated ovarioles showed lower intensity in RNase A-treated (Mean: 17.29; Min/Max gray values: 0/255; IntDen: 575.51) than in untreated controls (Mean: 29.39; Min/Max: 0/255; IntDen: 978.45).
Figure Legend Snippet: Immunolocalization of HEX 110 in RNase A-treated ovarioles (stage S2) of queenright nurse workers. (A) Control ovariole non-incubated with RNase A. HEX 110 green foci (labeled with anti-HEX 110/Alexa-Fluor 488) are abundant in the nucleus (arrowheads) and cytoplasm (arrows) of nurse (Nc) and follicle (Fc) cells surrounding the oocyte (stage 2, see Fig 2 ). (B) Ovariole incubated with RNase A shows scarce HEX 110 foci (arrows). DAPI-staining (blue) in B highlights the small nuclei of follicle cells (Fc) that surround the oocyte (stage 1, see Fig 2 ) and are interspersed between the large nurse cell (Nc) nuclei. Quantification of pixels intensity in the selected areas (dashed rectangles) of the images obtained from RNase A-treated and untreated ovarioles showed lower intensity in RNase A-treated (Mean: 17.29; Min/Max gray values: 0/255; IntDen: 575.51) than in untreated controls (Mean: 29.39; Min/Max: 0/255; IntDen: 978.45).

Techniques Used: Incubation, Labeling, Flow Cytometry, Staining

10) Product Images from "Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus"

Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

Journal: Scientific Reports

doi: 10.1038/s41598-018-26264-0

Isolation of Messenger Ribonucleoprotein Particles from  S. purpuratus  ova. Lysates of  S. purpuratus  ova were either untreated (−RNase) or treated with RNase A/T1 (+RNase) and applied to oligo-dT cellulose columns and the bound proteins analyzed by SDS-PAGE and visualized by staining with Coomassie brilliant blue (upper panel) or subjected to Western blotting with anti- Xenopus  YY1 antibody (lower panel). Fractions are indicated above the panel, L, load-on; U, unbound fraction; B, bound fraction. Positions of molecular mass markers and the YY1 band found upon Western blotting are indicated to the left of the panel.
Figure Legend Snippet: Isolation of Messenger Ribonucleoprotein Particles from S. purpuratus ova. Lysates of S. purpuratus ova were either untreated (−RNase) or treated with RNase A/T1 (+RNase) and applied to oligo-dT cellulose columns and the bound proteins analyzed by SDS-PAGE and visualized by staining with Coomassie brilliant blue (upper panel) or subjected to Western blotting with anti- Xenopus YY1 antibody (lower panel). Fractions are indicated above the panel, L, load-on; U, unbound fraction; B, bound fraction. Positions of molecular mass markers and the YY1 band found upon Western blotting are indicated to the left of the panel.

Techniques Used: Isolation, SDS Page, Staining, Western Blot

11) Product Images from "Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP (messenger ribonucleoprotein particle) complexes"

Article Title: Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP (messenger ribonucleoprotein particle) complexes

Journal:

doi: 10.1042/BJ20051548

RNA affinity purification of heparin-sepharose column fraction Heparin–sepharose fraction 3 was incubated with ARE-RNA-poly(A):oligo(dT) beads. Proteins were eluted from the RNA affinity matrix with a step gradient of NaCl. Eluted fractions were examined by gel-shift and UV cross-linking assays. ( A ) RNA gel-shift assays of column fractions. Aliquots of the fractions were incubated with 32 P-AREbcl -2 RNA in binding buffer and RNA–protein complexes were separated by electrophoresis on a 0.8% agarose gel. ( B ) Aliquots of the indicated fractions were separated by SDS/PAGE and detected by Coomassie Blue staining. ( C ) Aliquots of the fractions eluted with 0.4 and 0.6 M NaCl were incubated with 32 P-AREbcl - 2 RNA and exposed to UV light. Complexes were digested with RNase A and RNase T1 and proteins were separated by SDS/PAGE. Bands were detected by phosphorimaging of the SDS gel. MW, molecular mass markers; S, sample applied to the column; FT, flow through fraction; W, wash fraction. Arrows indicate the bands excised from the gel for MALDI-MS analysis.
Figure Legend Snippet: RNA affinity purification of heparin-sepharose column fraction Heparin–sepharose fraction 3 was incubated with ARE-RNA-poly(A):oligo(dT) beads. Proteins were eluted from the RNA affinity matrix with a step gradient of NaCl. Eluted fractions were examined by gel-shift and UV cross-linking assays. ( A ) RNA gel-shift assays of column fractions. Aliquots of the fractions were incubated with 32 P-AREbcl -2 RNA in binding buffer and RNA–protein complexes were separated by electrophoresis on a 0.8% agarose gel. ( B ) Aliquots of the indicated fractions were separated by SDS/PAGE and detected by Coomassie Blue staining. ( C ) Aliquots of the fractions eluted with 0.4 and 0.6 M NaCl were incubated with 32 P-AREbcl - 2 RNA and exposed to UV light. Complexes were digested with RNase A and RNase T1 and proteins were separated by SDS/PAGE. Bands were detected by phosphorimaging of the SDS gel. MW, molecular mass markers; S, sample applied to the column; FT, flow through fraction; W, wash fraction. Arrows indicate the bands excised from the gel for MALDI-MS analysis.

Techniques Used: Affinity Purification, Incubation, Electrophoretic Mobility Shift Assay, Binding Assay, Electrophoresis, Agarose Gel Electrophoresis, SDS Page, Staining, SDS-Gel, Flow Cytometry, Mass Spectrometry

RNA binding and UV cross-linking assays of heparin–sepharose column fractions ( A ) RNA gel-shift assay. Aliquots of the indicated fractions were incubated with 32 P-AREbcl -2 RNA in RNA binding buffer. Free and bound RNAs were separated by electrophoresis on a 0.8% agarose gel, which was dried and analysed by phosphorimaging. ( B ) and ( C ), aliquots of the indicated fractions were incubated with 32 P-AREbcl -2 RNA in RNA binding buffer and exposed to UV light for 30 min. Complexes were digested with RNase A and T1, and proteins were separated by SDS/PAGE. ( B ) Image of the Coomassie Blue stained gel. ( C ) Phosphorimage of the same SDS gel. MW, molecular mass markers; S, sample applied to the column; FT, flow through fraction; W, wash fraction.
Figure Legend Snippet: RNA binding and UV cross-linking assays of heparin–sepharose column fractions ( A ) RNA gel-shift assay. Aliquots of the indicated fractions were incubated with 32 P-AREbcl -2 RNA in RNA binding buffer. Free and bound RNAs were separated by electrophoresis on a 0.8% agarose gel, which was dried and analysed by phosphorimaging. ( B ) and ( C ), aliquots of the indicated fractions were incubated with 32 P-AREbcl -2 RNA in RNA binding buffer and exposed to UV light for 30 min. Complexes were digested with RNase A and T1, and proteins were separated by SDS/PAGE. ( B ) Image of the Coomassie Blue stained gel. ( C ) Phosphorimage of the same SDS gel. MW, molecular mass markers; S, sample applied to the column; FT, flow through fraction; W, wash fraction.

Techniques Used: RNA Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Electrophoresis, Agarose Gel Electrophoresis, SDS Page, Staining, SDS-Gel, Flow Cytometry

12) Product Images from "Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA"

Article Title: Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA

Journal: Cellular and Molecular Life Sciences

doi: 10.1007/s00018-015-1908-0

Summary of the enzymatic and chemical probing results for  IFNA5  RNA (197–446). Mapping data are depicted upon common predicted secondary structure of different species using Centroid program  http://www.ncrna.org/centroidfold/ . Enzymatic cuts are showed as  arrow heads  (RNase T1),  circles  (RNase V1) and  arrows  (RNase A). The bases modified by DEPC are indicated by  rhomboids  and those cleaved by Pb 2+  by  stars . Strong cleavages are indicated by  filled symbols  and moderate ones by  unfilled symbols . (+) and (−) enhanced or protected cleavage in MgCl 2 .  Numbers  indicate nucleotide positions.  D  domain,  l  loop,  PK  possible pseudoknot. In  blue , sequences participating in a predicted pseudoknot. Highlighted in  gray  are CAR-E sequences
Figure Legend Snippet: Summary of the enzymatic and chemical probing results for IFNA5 RNA (197–446). Mapping data are depicted upon common predicted secondary structure of different species using Centroid program http://www.ncrna.org/centroidfold/ . Enzymatic cuts are showed as arrow heads (RNase T1), circles (RNase V1) and arrows (RNase A). The bases modified by DEPC are indicated by rhomboids and those cleaved by Pb 2+ by stars . Strong cleavages are indicated by filled symbols and moderate ones by unfilled symbols . (+) and (−) enhanced or protected cleavage in MgCl 2 . Numbers indicate nucleotide positions. D domain, l loop, PK possible pseudoknot. In blue , sequences participating in a predicted pseudoknot. Highlighted in gray are CAR-E sequences

Techniques Used: Modification

Enzymatic probing of the secondary structure of  IFNA5  RNA (197–446).  a ,  b  5′-[ 32 P] end-labelled RNA.  b ,  c  3′-[ 32 P] end-labelled RNA. For  a  and  b lane 1  is the RNA maintained on ice ( I );  lane 2  alkaline hydrolysis reaction ( OH );  lane 3  RNase T1 reaction under denaturing conditions ( T1L );  lane 4  RNase T1 ( T1 ),  lane 5  RNase V1 ( V1 ) and  lane 6  RNase A ( A ) under standard conditions, respectively. For  c  and  d  products digested with alkali ( OH ), RNase T1 under denaturing conditions ( T1L ), RNase T1 ( T1 ), RNase V1 ( V1 ), RNase A ( A ) and  E. coli  RNase 1 ( 1 ) under standard conditions were analysed in  lanes 1  to  6 , respectively. Denaturing gels were at 10 ( a ), 6 ( b  and  d ) and 15 % ( c ) polyacrylamide. The  numbers  on the  right  indicate the point of digestion cleaved by RNase T1 under denaturing conditions ( T1L ), as identified with the help of the OH sequence ladders run on the  left
Figure Legend Snippet: Enzymatic probing of the secondary structure of IFNA5 RNA (197–446). a , b 5′-[ 32 P] end-labelled RNA. b , c 3′-[ 32 P] end-labelled RNA. For a and b lane 1 is the RNA maintained on ice ( I ); lane 2 alkaline hydrolysis reaction ( OH ); lane 3 RNase T1 reaction under denaturing conditions ( T1L ); lane 4 RNase T1 ( T1 ), lane 5 RNase V1 ( V1 ) and lane 6 RNase A ( A ) under standard conditions, respectively. For c and d products digested with alkali ( OH ), RNase T1 under denaturing conditions ( T1L ), RNase T1 ( T1 ), RNase V1 ( V1 ), RNase A ( A ) and E. coli RNase 1 ( 1 ) under standard conditions were analysed in lanes 1 to 6 , respectively. Denaturing gels were at 10 ( a ), 6 ( b and d ) and 15 % ( c ) polyacrylamide. The numbers on the right indicate the point of digestion cleaved by RNase T1 under denaturing conditions ( T1L ), as identified with the help of the OH sequence ladders run on the left

Techniques Used: Sequencing

13) Product Images from "Ai-lncRNA EGOT enhancing autophagy sensitizes paclitaxel cytotoxicity via upregulation of ITPR1 expression by RNA-RNA and RNA-protein interactions in human cancer"

Article Title: Ai-lncRNA EGOT enhancing autophagy sensitizes paclitaxel cytotoxicity via upregulation of ITPR1 expression by RNA-RNA and RNA-protein interactions in human cancer

Journal: Molecular Cancer

doi: 10.1186/s12943-019-1017-z

EGOT regulates ITPR1 expression in cis and in trans in human cancer. a Nuclear and cytoplasmic fractions of HeLa and T47D cells were subjected to qRT-PCR. U1 is the nuclear (Nul) positive control; GAPDH is the cytoplasmic (Cyt) positive control. b RNA-FISH performed in HeLa and T47D cells. EGOT probes are red; ACTIN probes are green; and ACTIN served as the positive control (1000 × magnification). c , d Expression of pre-ITPR1 in EGOT overexpression and knockdown cells by qRT-PCR. e Combined RNase resistance and dual-RNA-FISH analysis. Before hybridization, T47D cells were treated with RNase A or RNase III. Hybridization was performed with specific probes against EGOT and ITPR1 transcripts. Nuclei are stained with DAPI. EGOT probes are red; ITPR1 probes are yellow; and ACTIN is the positive control. Arrows indicate foci (1000 × magnification). f Domain mapping of the EGOT transcript. g , h Expression of pre-ITPR1 and ITPR1 mRNA ( g ) and protein ( h ) in MCF7 cells transfected with different fragments of EGOT lentivirus. i An RNA pull-down assay was conducted using the Flag-MS2bp-MS2bs-based system followed by western blotting of lysates from MDA-MB-231, T47D, and UACC-812 cells after transfection with MS2- EGOT and MS2-vector (control). j Antibodies against hnRNPH1 were used for RIP, followed by qRT-PCR, in MCF7 and MDA-MB-231 cells. k, l ITPR1 mRNA expression was analyzed by qRT-PCR after hnRNPH1 knockdown via siRNAs ( k ), followed by western blotting ( l ). m The RNA pull-down assay was conducted using the Flag-MS2bp-MS2bs-based system in T47D cells after transfection with MS2- EGOT lentivirus along with different fragments of EGOT lentivirus, followed by western blotting. n Western blot of hnRNPH1 pulled down by F2-MS2 and mutated F2 RNA in 293 T cells. The red underlined sequences indicate the potential binding sites that were mutated into Us in F2, while the A, B and C binding sites were mutated at the same time to F2-mut ABC. Data are shown as the mean ± s.d. Student’s t-test was used for statistical analysis: * P
Figure Legend Snippet: EGOT regulates ITPR1 expression in cis and in trans in human cancer. a Nuclear and cytoplasmic fractions of HeLa and T47D cells were subjected to qRT-PCR. U1 is the nuclear (Nul) positive control; GAPDH is the cytoplasmic (Cyt) positive control. b RNA-FISH performed in HeLa and T47D cells. EGOT probes are red; ACTIN probes are green; and ACTIN served as the positive control (1000 × magnification). c , d Expression of pre-ITPR1 in EGOT overexpression and knockdown cells by qRT-PCR. e Combined RNase resistance and dual-RNA-FISH analysis. Before hybridization, T47D cells were treated with RNase A or RNase III. Hybridization was performed with specific probes against EGOT and ITPR1 transcripts. Nuclei are stained with DAPI. EGOT probes are red; ITPR1 probes are yellow; and ACTIN is the positive control. Arrows indicate foci (1000 × magnification). f Domain mapping of the EGOT transcript. g , h Expression of pre-ITPR1 and ITPR1 mRNA ( g ) and protein ( h ) in MCF7 cells transfected with different fragments of EGOT lentivirus. i An RNA pull-down assay was conducted using the Flag-MS2bp-MS2bs-based system followed by western blotting of lysates from MDA-MB-231, T47D, and UACC-812 cells after transfection with MS2- EGOT and MS2-vector (control). j Antibodies against hnRNPH1 were used for RIP, followed by qRT-PCR, in MCF7 and MDA-MB-231 cells. k, l ITPR1 mRNA expression was analyzed by qRT-PCR after hnRNPH1 knockdown via siRNAs ( k ), followed by western blotting ( l ). m The RNA pull-down assay was conducted using the Flag-MS2bp-MS2bs-based system in T47D cells after transfection with MS2- EGOT lentivirus along with different fragments of EGOT lentivirus, followed by western blotting. n Western blot of hnRNPH1 pulled down by F2-MS2 and mutated F2 RNA in 293 T cells. The red underlined sequences indicate the potential binding sites that were mutated into Us in F2, while the A, B and C binding sites were mutated at the same time to F2-mut ABC. Data are shown as the mean ± s.d. Student’s t-test was used for statistical analysis: * P

Techniques Used: Expressing, Quantitative RT-PCR, Positive Control, Fluorescence In Situ Hybridization, Over Expression, Hybridization, Staining, Transfection, Pull Down Assay, Western Blot, Multiple Displacement Amplification, Plasmid Preparation, Binding Assay

14) Product Images from "Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum"

Article Title: Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum

Journal: Journal of Extracellular Vesicles

doi: 10.3402/jev.v3.24783

Characterization of FBS-derived extracellular vesicles. FBS-derived vesicles were collected at 120,000×g for 18 hours, dissolved in PBS and re-pelleted at 120,000×g for 70 minutes.  a)  To visualize if FBS had vesicular structures present, TEM was performed on the EV-enriched pellet. The scale bar represents 200 nm.  b)  Immuno-blotting for the exosome enriched proteins; TSG101, CD81, and CD63. Pellet of EVs were obtained from different batch of FBS. Cell lysate from human mast cells (HMC-1) and mouse fibroblast (NIH-3T3), as well as HMC-1 derived exosomes were used as positive controls. One hundred microgram of protein was loaded per well for all samples.  c)  RNA was isolated using the miRCURY total RNA isolation kit and quantified with a Bioanalyzer.  d)  The FBS-derived EV-enriched pellet was incubated with RNase A (0.5 µg/µl) alone  (i)  or was incubated with proteinase K (0.05 µg/µl) prior to the RNase treatment  (ii)  to study mode of RNA protection. Cellular RNA was used as control for the RNase activity  (iii) . Graphs are representative of the RNA profile obtained with a Bioanalyzer from two independent experiments.  e)  PKH67-labelled FBS-derived EVs were incubated with 7×10 4 cells (A549) for 6 or 20 hours and visualized with a fluorescent microscope (40X). PBS was used as control for determining the PKH67 background labelling.
Figure Legend Snippet: Characterization of FBS-derived extracellular vesicles. FBS-derived vesicles were collected at 120,000×g for 18 hours, dissolved in PBS and re-pelleted at 120,000×g for 70 minutes. a) To visualize if FBS had vesicular structures present, TEM was performed on the EV-enriched pellet. The scale bar represents 200 nm. b) Immuno-blotting for the exosome enriched proteins; TSG101, CD81, and CD63. Pellet of EVs were obtained from different batch of FBS. Cell lysate from human mast cells (HMC-1) and mouse fibroblast (NIH-3T3), as well as HMC-1 derived exosomes were used as positive controls. One hundred microgram of protein was loaded per well for all samples. c) RNA was isolated using the miRCURY total RNA isolation kit and quantified with a Bioanalyzer. d) The FBS-derived EV-enriched pellet was incubated with RNase A (0.5 µg/µl) alone (i) or was incubated with proteinase K (0.05 µg/µl) prior to the RNase treatment (ii) to study mode of RNA protection. Cellular RNA was used as control for the RNase activity (iii) . Graphs are representative of the RNA profile obtained with a Bioanalyzer from two independent experiments. e) PKH67-labelled FBS-derived EVs were incubated with 7×10 4 cells (A549) for 6 or 20 hours and visualized with a fluorescent microscope (40X). PBS was used as control for determining the PKH67 background labelling.

Techniques Used: Derivative Assay, Transmission Electron Microscopy, Isolation, Incubation, Activity Assay, Microscopy

15) Product Images from "A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop"

Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks802

Hybrid G-quadruplexes form during transcription and are dependent on CSB II. ( A )  In vitro  transcription using the mitochondrial transcription apparatus on supercoiled templates containing either wildtype or mutant CSB II. Transcription reactions were divided into three parts, and either not treated or treated with RNase A and/or hRNaseH1, as indicated. RNase A-treatment reveals a non-degradable product of 45–50 bp (marked by asterisks) that is dependent on the CSB II sequence and Hoogsteen base pairing. ( B ) The labeled CSB II RNA oligonucleotide was allowed to form G-quadruplexes alone (lanes 1–3), with wildtype CSB II DNA oligonucleotide of identical sequence (lanes 4–6), or with a reverse complement DNA oligonucleotide (lanes 7–9), and then subjected to treatment with RNase A or hRNaseH1 in order to show that the G-quadruplex hybrid is resistant to hRNaseH1 (lane 6). The Watson–Crick basepaired hybrid with reverse complement DNA was degraded by hRNaseH1 as expected (lane 9). M, marker lane. ( C ) Transcription on templates encompassing base positions 1–477 of human mtDNA and containing either dGTP or 7-deaza-dGTP (lanes 1 and 2). After transcription, part of each reaction was treated with RNaseA (lanes 3 and 4). The RNaseA-resistant species of ∼50 bp (indicated by black bar) are absent when G-quadruplex formation with the template is eliminated (lane 3). M, marker lane.
Figure Legend Snippet: Hybrid G-quadruplexes form during transcription and are dependent on CSB II. ( A ) In vitro transcription using the mitochondrial transcription apparatus on supercoiled templates containing either wildtype or mutant CSB II. Transcription reactions were divided into three parts, and either not treated or treated with RNase A and/or hRNaseH1, as indicated. RNase A-treatment reveals a non-degradable product of 45–50 bp (marked by asterisks) that is dependent on the CSB II sequence and Hoogsteen base pairing. ( B ) The labeled CSB II RNA oligonucleotide was allowed to form G-quadruplexes alone (lanes 1–3), with wildtype CSB II DNA oligonucleotide of identical sequence (lanes 4–6), or with a reverse complement DNA oligonucleotide (lanes 7–9), and then subjected to treatment with RNase A or hRNaseH1 in order to show that the G-quadruplex hybrid is resistant to hRNaseH1 (lane 6). The Watson–Crick basepaired hybrid with reverse complement DNA was degraded by hRNaseH1 as expected (lane 9). M, marker lane. ( C ) Transcription on templates encompassing base positions 1–477 of human mtDNA and containing either dGTP or 7-deaza-dGTP (lanes 1 and 2). After transcription, part of each reaction was treated with RNaseA (lanes 3 and 4). The RNaseA-resistant species of ∼50 bp (indicated by black bar) are absent when G-quadruplex formation with the template is eliminated (lane 3). M, marker lane.

Techniques Used: In Vitro, Mutagenesis, Sequencing, Labeling, Marker

A longer R-loop can form during transcription of human mtDNA with phage T7 RNA polymerase. ( A ) Transcription of a human mtDNA template containing wildtype or mutant CSB II with T7 RNA pol in the presence of either GTP (lanes 1–3 and 7–9) or 7-deaza-GTP (lanes 4–6 and 10–12). A hybrid species of ∼120 bp is revealed upon RNase A treatment (lane 2) and is sensitive to hRNaseH1 (lane 3). This longer hybrid is dependent on CSB II and not observed in the presence of 7-deaza-GTP (lanes 4–6). ( B ) Schematic presentation of the RNA–DNA hybrid G-quadruplex that forms between the RNA and the non-template DNA strand during transcription of mtDNA. Under some conditions, an extended R-loop may be formed, similar to that reported in (  22 ).
Figure Legend Snippet: A longer R-loop can form during transcription of human mtDNA with phage T7 RNA polymerase. ( A ) Transcription of a human mtDNA template containing wildtype or mutant CSB II with T7 RNA pol in the presence of either GTP (lanes 1–3 and 7–9) or 7-deaza-GTP (lanes 4–6 and 10–12). A hybrid species of ∼120 bp is revealed upon RNase A treatment (lane 2) and is sensitive to hRNaseH1 (lane 3). This longer hybrid is dependent on CSB II and not observed in the presence of 7-deaza-GTP (lanes 4–6). ( B ) Schematic presentation of the RNA–DNA hybrid G-quadruplex that forms between the RNA and the non-template DNA strand during transcription of mtDNA. Under some conditions, an extended R-loop may be formed, similar to that reported in ( 22 ).

Techniques Used: Mutagenesis

16) Product Images from "Improved Loading of Plasma-Derived Extracellular Vesicles to Encapsulate Antitumor miRNAs"

Article Title: Improved Loading of Plasma-Derived Extracellular Vesicles to Encapsulate Antitumor miRNAs

Journal: Molecular Therapy. Methods & Clinical Development

doi: 10.1016/j.omtm.2019.01.001

miRNA Loading in EVs and Protection from Enzymatic Degradation EVs were engineered with an Alexa Fluor 555-labeled control miRNA (miR) to perform FACS analysis. (A) miRNA enrichment in EVs was measured as percentage of signal intensity in EVs electroporated or incubated with miRNA (EVe+miR and EVi+miR, respectively) in comparison with controls, naive EVs, and vesicles electroporated alone (EVe) (n = 3). ANOVA with Turkey’s multiple-comparisons test. (B) EVs electroporated or incubated with the miRNA were used to treat target TECs for 24 h (4.2 × 10 9 EVs/mL). Engineered miRNA transfer was measured as miRNA percentage signal intensity in untreated cells (CTL−), cells treated with control EVs, vesicles electroporated alone (EVe), and EVs electroporated or incubated with miRNA (EVe+miR and EVi+miR, respectively) (n = 5). ANOVA with Dunnett’s multiple-comparisons test versus EVe+miR. (C) EVs were engineered with different doses of miRNA cel-39 (5, 10, and 20 pmol), and miRNA loading was analyzed in qRT-PCR experiments. Data are represented as ln(RQ) of miRNA cel-39 in comparison with control EVs (n = 3). (D) Engineered EVs with miRNA cel-39 were treated with RNase A for 30 min at 37°C. miRNA protection was evaluated using qRT-PCR analysis as percentage of miRNA expression (ΔCt values) after RNase degradation in comparison with untreated samples. EVs engineered with 10 or 20 pmol miRNA dose were tested, and the different effect of electroporation and incubation was analyzed (n = 3). ANOVA with Turkey’s multiple-comparisons test. (E) EVs incubated or electroporated with miRNA cel-39 were treated or not with RNase and analyzed for their small RNA profile using Bioanalyzer in comparison with each control EV sample. miRNA enrichment was expressed as percentage of miRNA on total small RNA content. ANOVA with Dunnett’s multiple-comparisons test (n = 3). Data are expressed as mean ± SEM. *p
Figure Legend Snippet: miRNA Loading in EVs and Protection from Enzymatic Degradation EVs were engineered with an Alexa Fluor 555-labeled control miRNA (miR) to perform FACS analysis. (A) miRNA enrichment in EVs was measured as percentage of signal intensity in EVs electroporated or incubated with miRNA (EVe+miR and EVi+miR, respectively) in comparison with controls, naive EVs, and vesicles electroporated alone (EVe) (n = 3). ANOVA with Turkey’s multiple-comparisons test. (B) EVs electroporated or incubated with the miRNA were used to treat target TECs for 24 h (4.2 × 10 9 EVs/mL). Engineered miRNA transfer was measured as miRNA percentage signal intensity in untreated cells (CTL−), cells treated with control EVs, vesicles electroporated alone (EVe), and EVs electroporated or incubated with miRNA (EVe+miR and EVi+miR, respectively) (n = 5). ANOVA with Dunnett’s multiple-comparisons test versus EVe+miR. (C) EVs were engineered with different doses of miRNA cel-39 (5, 10, and 20 pmol), and miRNA loading was analyzed in qRT-PCR experiments. Data are represented as ln(RQ) of miRNA cel-39 in comparison with control EVs (n = 3). (D) Engineered EVs with miRNA cel-39 were treated with RNase A for 30 min at 37°C. miRNA protection was evaluated using qRT-PCR analysis as percentage of miRNA expression (ΔCt values) after RNase degradation in comparison with untreated samples. EVs engineered with 10 or 20 pmol miRNA dose were tested, and the different effect of electroporation and incubation was analyzed (n = 3). ANOVA with Turkey’s multiple-comparisons test. (E) EVs incubated or electroporated with miRNA cel-39 were treated or not with RNase and analyzed for their small RNA profile using Bioanalyzer in comparison with each control EV sample. miRNA enrichment was expressed as percentage of miRNA on total small RNA content. ANOVA with Dunnett’s multiple-comparisons test (n = 3). Data are expressed as mean ± SEM. *p

Techniques Used: Labeling, FACS, Incubation, CTL Assay, Quantitative RT-PCR, Expressing, Electroporation

17) Product Images from "Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers"

Article Title: Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers

Journal: Nature cell biology

doi: 10.1038/ncb1800

Glioblastoma cells produce microvesicles containing RNA Scanning EM image of a primary glioblastoma cell (bar = 10 μm). (b) Higher magnification showing the microvesicles on the cell surface. Vesicles can be binned into diameters of around 50 nm and 500 nm (bar = 1 μm). (c) Microvesicles were exposed to RNase A or mock-treated prior to RNA isolation and levels of RNA determined (n = 5). (d) Bioanalyzer data shows the size distribution of total RNA extracted from primary glioblastoma cells and (e) microvesicles isolated from them. The smallest peak represents an internal standard. The two prominent peaks in (d) (arrows) represent 18S (left) and 28S (right) ribosomal RNA, absent in microvesicles.
Figure Legend Snippet: Glioblastoma cells produce microvesicles containing RNA Scanning EM image of a primary glioblastoma cell (bar = 10 μm). (b) Higher magnification showing the microvesicles on the cell surface. Vesicles can be binned into diameters of around 50 nm and 500 nm (bar = 1 μm). (c) Microvesicles were exposed to RNase A or mock-treated prior to RNA isolation and levels of RNA determined (n = 5). (d) Bioanalyzer data shows the size distribution of total RNA extracted from primary glioblastoma cells and (e) microvesicles isolated from them. The smallest peak represents an internal standard. The two prominent peaks in (d) (arrows) represent 18S (left) and 28S (right) ribosomal RNA, absent in microvesicles.

Techniques Used: Electron Microscopy, Isolation

18) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00428

Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

Techniques Used: Activation Assay

RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

Techniques Used: Labeling, Injection, Mouse Assay

RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

Techniques Used:

Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

Techniques Used: Staining

Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

Techniques Used: Immunostaining, Staining

RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

Techniques Used:

RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

Techniques Used: Immunostaining, Marker, Staining

19) Product Images from "Fabrication of Stable and RNase-Resistant RNA Nanoparticles Active in Gearing the Nanomotors for Viral DNA-Packaging"

Article Title: Fabrication of Stable and RNase-Resistant RNA Nanoparticles Active in Gearing the Nanomotors for Viral DNA-Packaging

Journal:

doi: 10.1021/nn1024658

Assembly activities of the 2’-F-C  U pRNA Aa’ and its non-modifed counterparts in the presence or absence of RNase A.
Figure Legend Snippet: Assembly activities of the 2’-F-C U pRNA Aa’ and its non-modifed counterparts in the presence or absence of RNase A.

Techniques Used:

Urea-PAGE denatured gel showing the stability between non-modified pRNA Aa’ and 2’-F-C U pRNA Aa’ after incubation at different time point in the presence of (A) RNase A (1 mg/ml), and (B) fetal bovine serum (10%).
Figure Legend Snippet: Urea-PAGE denatured gel showing the stability between non-modified pRNA Aa’ and 2’-F-C U pRNA Aa’ after incubation at different time point in the presence of (A) RNase A (1 mg/ml), and (B) fetal bovine serum (10%).

Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Incubation

20) Product Images from "RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway"

Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00428

Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P
Figure Legend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

Techniques Used: Acetylene Reduction Assay, Staining, Microscopy

RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P
Figure Legend Snippet: RNase A induces NPC proliferation through the ERK pathway. (A) At 1 DIV, dissociated cortical and hippocampal cultures were treated with 100 μg/ml RNase A (Qiagen) and harvested at different time-points, as indicated. ERK activities were detected by means of immunoblotting with antibody recognizing phosphorylated ERK1/2 (pERK). (B) Pretreatment with U0126 (a MEK1/2 inhibitor) at dosages of 0, 5, or 10 μM for 30 min was performed to examine the specificity of RNase A for ERK activation. RNase A or BSA control (100 μg/ml) was added 20 min before harvesting. Quantification data shown at the bottoms of (A) and (B) are mean and SEM of three independent experiments. Statistical analyses were performed using one-way ANOVA (A) and two-way ANOVA (B) . ** P

Techniques Used: Activation Assay

RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P
Figure Legend Snippet: RNase A treatment induces EdU incorporation in mouse brains. (A) Schematic timeline for RNase A (Qiagen) treatment and EdU labeling. Intracerebroventricular (icv) injection of 180 μg RNase A or BSA control was performed once per day for one to four days, as indicated. After the last injection of each group, mice received a single intraperitoneal (i.p.) injection of EdU (100 mg/kg) to label proliferated cells. Mouse brains were harvested at day 8 after the first icv injection. (B) Schematic diagram showing the position of the icv injection. * indicates the non-injected side. (C) Representative images of EdU labeling of the BSA x4 and RNase A x4 groups in the subventricular region of the lateral ventricle (SVZ) and hippocampus. Images in the middle panel of (C) are enlargements of the squares in the respective upper panel; scale bar, 1 mm. Arrow points a EdU-positive cell at subgranular zone of dentate gyrus. Bottom panel of (C) ; images (i, ii: SVZ; iii, v: zone CA3 of hippocampus; iv, vi: dentate gyrus, DG) are enlargements of the squares in the middle panels; scale bar, 200 μm. (D–G) Quantification of EdU-positive cells in both sides of the (D, F) lateral ventricle and (E, G) hippocampus. The same datasets of RNase A x4 are used in (D, F) and (E, G) . Data represent mean ± SD ( n = 4 mice per group). * P

Techniques Used: Labeling, Injection, Mouse Assay

RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.
Figure Legend Snippet: RNase A-induced NPCs migrate to various brain regions. (A) Schematic timeline for RNase A (Qiagen) injection into lateral ventricles and BrdU labeling in vivo . (B) BrdU staining 30 days after the first BSA or RNase A injection. Upper, BSA group; lower, RNase A group. (C) Double immunostaining with BrdU and Nestin or GFAP antibodies. Counter-staining with DAPI was performed. The results for the amygdala and hippocampal CA1 region are shown. Note that Nestin was concentrated at the nuclei of migrating NPCs. White arrows indicate some double-positive cells. Scale bars, (B) 1 mm; (C) 20 μm.

Techniques Used: Injection, Labeling, In Vivo, BrdU Staining, Double Immunostaining, Staining

Dividing NPCs are present in neuronal cultures.  (A)  SOX2 + Nestin +  cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive.  (B)  Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars,  (A)  50 μm;  (B)  20 μm.
Figure Legend Snippet: Dividing NPCs are present in neuronal cultures. (A) SOX2 + Nestin + cells are present in neuronal cultures. RNase A-induced Nestin-positive cells are also SOX2-positive. (B) Live recording of neuronal culture from DIV 0 to 4. The video is available as Movie S1. Bright-field images at the indicated time-points are shown. Asterisks indicate NPCs or their daughter cells. Asterisks of the same color indicate the same lineage of cells. Black asterisks at DIV 0 and 1 indicate two cells, which were dead at DIV1. Scale bars, (A) 50 μm; (B) 20 μm.

Techniques Used:

Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P
Figure Legend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

Techniques Used: Staining

Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P
Figure Legend Snippet: Dosage effect of RNase A on NPC proliferation. Different amounts (25, 50, 100 μg/ml) of Invitrogen RNase A were added to mouse cortex and hippocampus neuronal cultures at 1 DIV and grown for 3 days. BSA (100 μg/ml) was included as a control. BrdU was added to cultures 2 h before harvesting. Immunostaining was performed with BrdU and Nestin antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. (B) Quantifications of the percentage of BrdU + cells (upper) and Nestin + cells (bottom) in total cell number. Data represent mean plus SD. The experiments were independently repeated four times. Scale bar, 50 μm. Statistical analyses were performed using one-way ANOVA. * P

Techniques Used: Immunostaining, Staining

RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P
Figure Legend Snippet: RNase A treatment promotes the growth of neurospheres. (A) Photographs of primary neurospheres treated with RNase A (Invitrogen, 25, 50, and 100 μg/ml) and grown for 9 days in 96-well plates. The medium did not contain the typical growth factors, such as EGF and FGF2, for NPCs. Scale bar, 300 μm. (B) Quantification of averaged area of each neurosphere colony in the photographs. The experiments were independently repeated four times. Mean and SD are shown. Statistical analyses were performed using one-way ANOVA. * P

Techniques Used:

RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P
Figure Legend Snippet: RNase A treatment increases numbers of NPCs in dissociated neuronal cultures. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml BSA or Invitrogen RNase A at 1 DIV and grown for 3 days. (A) Representative images of immunostaining with Nestin, an NPC marker, are shown. Counter-staining with DAPI was performed to label cell nuclei. The number of DAPI + cells represents the total cell number. (B) Quantifications, including the number of total DAPI + cells, the number of Nestin + cells and the percentage of Nestin + cells in total DAPI + cells. Mean and SD of three independent experiments are shown. Scale bars, 50 μm. Statistical analyses were performed using unpaired t -tests. ** P

Techniques Used: Immunostaining, Marker, Staining

Related Articles

Diagnostic Assay:

Article Title: Identification of a Divergent Lineage Porcine Pestivirus in Nursing Piglets with Congenital Tremors and Reproduction of Disease following Experimental Inoculation
Article Snippet: Varied porcine tissues (serum, cerebrum, cerebellum, spinal cord, cerebrospinal fluid (CSF), and/or lung) from three diagnostic investigations of CT were obtained: lung tissue from a single piglet (ID 20130103); either pooled brain tissue or pooled lung tissue from six piglets (ID 20120705); and CSF (n = 2; Farm B), serum (n = 2; Farm A and B), and lung (n = 2; Farm A and B) from six different piglets originating from two different farms (ID 2014016573). .. Serum or tissue homogenates were re-suspended in Hanks balanced salt solution (Corning-Cellgro) and enriched for viral particle protected nucleic acids by digestion with a combination of nucleases: RNase A (Invitrogen), Baseline Zero DNase (Epicentre), and Turbo DNase (Invitrogen) as described previously [ ].

Centrifugation:

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: Cell pellets containing approximately one million crosslinked cells were resuspended in cold lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.2 % NP-40) and incubated on ice for 10 min. .. Nuclei were pelleted at 2500g for 60 s, resuspended in 100 μL of 0.5× DNase I digestion buffer [0.5× DNase I digestion buffer (Thermo), 0.5 mM MnCl2 ] containing 0.2 % SDS, and incubated at 37 °C for 30 min. An equal volume of 0.5× DNase I digestion buffer containing 2 % Triton X-100 and 4 U RNase A (Thermo) was added and incubation at 37 °C was continued for 10 min. Then, 1.5 U DNase I (Thermo) was added and digestion carried out at room temperature for 4 min. DNase I digestion was stopped by adding 40 μL of 6× Stop Solution (125 mM EDTA, 2.5 % SDS), followed by centrifugation at 2500g for 60 s. Nuclei were resuspended in 150 μL nuclease-free H2 O (Ambion), and purified with two volumes (300 μL) of AMPure XP SPRI magnetic beads (Beckman Coulter). .. The resulting mixture was well mixed, incubated at room temperature for 5 min, collected via DynaMag-Spin magnet (Invitrogen), washed twice with 80 % ethanol, and air dried for 2 min.

Article Title: Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines
Article Snippet: Cell pellets of TMZ-treated and untreated samples were solubilised in 0.1 ml 40 mM Tris supplemented with 20 μg/ml RNase A (bovine pancreas source; USB Corporation), 50 U/ml DNase I (bovine pancreas source; USB Corporation), complete ethylenediamine tetraacetic acid (EDTA)-free protease inhibitors (Roche Diagnostics) and 0.5 mM phenylmethylsulphonylfluoride (PMSF) (USB Corporation), and lysed by vortexing. .. After centrifugation at 16,000g , the pellets were resuspended by vortexing in 0.1 ml extraction solution containing 8 M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), 40 mM Tris, 0.2% carrier ampholytes and 2 mM tributyl phosphine (these reagents were included in ReadyPrep sequential extraction kit; Bio-Rad, Hercules, CA, USA).

Article Title: HIV-1 remodels the nuclear pore complex
Article Snippet: Pellets were resuspended in 1.8 M SHKM and were underlayed with 2.2 M SHKM for centrifugation at 25,000 rpm for 2.5 h at 4°C. .. Pellets were resuspended in 0.25 M SHKM (50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ), treated with RNase A and DNase I (Invitrogen) for 15 min, and resuspended in 10% SHM (0.3 M sucrose, 10 mM Hepes, pH 7.4, and 2 mM MgCl2 ) for centrifugation at 5,000 rpm for 10 min at 4°C. .. Pellets were resuspended in 10% SHM, again treated with RNase A and DNase I for 15 min, underlayed with 30% SHM, and centrifuged at 5,000 rpm for 30 min at 4°C.

Polymerase Chain Reaction:

Article Title: DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection
Article Snippet: For analysis of the tRNA-bound MLV nucleic acid, the same procedure was used, except that the eluted nucleic acid was reverse transcribed prior to PCR with the PR primer and another primer that annealed to nucleotides (nt) 39 to 57 in tRNAPro (PtRNA in ) (5′-GCTCTCCAGGGCCCAAGTT-3′) ( ). .. For the nuclease treatments, after the nucleic acids were released from the protein cross-link, they were ethanol precipitated and treated at 37°C with 50 U RNase A (Thermo) for 20 min in the presence of 300 mM NaCl, 4 U DNase I (Roche) with the reaction buffer provided with the enzyme for 20 min, or 3 U of RNase H (Thermo) for 20 min in the reaction buffer provided with the enzyme.

Construct:

Article Title: Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications
Article Snippet: All UNG constructs were expressed in the E. coli BL21(DE3) ung-151 strain and purified using Ni-NTA affinity resin (Qiagen, Hilden, Germany). .. Cells were harvested and lyzed in lysis buffer (50 mM TRIS·HCl, pH = 8.0, 300 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1×cOmplete ULTRA™ EDTA free protease inhibitor cocktail tablet (Roche), 0.1 mg/ml lysozyme, 0.1 mg/ml DNase (Sigma, St. Louis, MO, USA) and 0.01 mg/ml RNAse A (Invitrogen, Carlsbad, CA, USA)) assisted with sonication.

SYBR Green Assay:

Article Title: A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA
Article Snippet: After 20 min, Heparin (20 IE/ml) or Hirudin (20 µg/ml) were added in the same concentration as used for stimulation (see above) for 40 min, followed by DNase I (Fermentas, St. Leon Rot, Germany) or RNase A (Fermentas, St. Leon Rot, Germany) digestion at 37°C (CpG-ODN/pArg 4 U DNase I 4 h; CpG-ODN/DOTAP 3 U DNase I 4 h; 9.2 s-RNA/DOTAP 250 ng RNase A 10 min; 3p-dsRNA/Lipofectamine 500 ng RNase A 90 min). .. After 20 min, Heparin (20 IE/ml) or Hirudin (20 µg/ml) were added in the same concentration as used for stimulation (see above) for 40 min, followed by DNase I (Fermentas, St. Leon Rot, Germany) or RNase A (Fermentas, St. Leon Rot, Germany) digestion at 37°C (CpG-ODN/pArg 4 U DNase I 4 h; CpG-ODN/DOTAP 3 U DNase I 4 h; 9.2 s-RNA/DOTAP 250 ng RNase A 10 min; 3p-dsRNA/Lipofectamine 500 ng RNase A 90 min).

Incubation:

Article Title: Induction of chondrogenesis of human placenta-derived mesenchymal stem cells via heparin-grafted human fibroblast derived matrix
Article Snippet: WI-38 human lung fibroblasts (ATCC, CCL-75) were cultured at the cell density of 2 × 104 cells/cm2 on the tissue culture dish (100 mm diameter) for 7 days in the DMEM supplemented with 10% FBS and 1% P/S. .. Once confluent, cell-loaded culture dish was washed twice with PBS, incubated briefly in a detergent solution containing 0.15% Triton X-100 (AMRESCO, Inc., Dallas, USA) and 10 mM NH4 OH (Sigma; St. Louis, MO, USA) at 37 °C, and then treated with 50 U/mL DNase I and 50 μg/mL RNase A (Invitrogen) for 1 h. After the decellularization process, ECMs were collected into centrifuge tubes and stored at 4 °C for future usage. .. hFDM was washed with PBS and saturated with 0.05 M 2-(Nmorpholino) ethanesulfonic acid hydrate (MES) buffer (pH = 5.5) (M2933, Sigma).

Article Title: A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA
Article Snippet: CpG 2006, 9.2 s and 3p-dsRNA were incubated with corresponding delivery agent as described above. .. After 20 min, Heparin (20 IE/ml) or Hirudin (20 µg/ml) were added in the same concentration as used for stimulation (see above) for 40 min, followed by DNase I (Fermentas, St. Leon Rot, Germany) or RNase A (Fermentas, St. Leon Rot, Germany) digestion at 37°C (CpG-ODN/pArg 4 U DNase I 4 h; CpG-ODN/DOTAP 3 U DNase I 4 h; 9.2 s-RNA/DOTAP 250 ng RNase A 10 min; 3p-dsRNA/Lipofectamine 500 ng RNase A 90 min).

Article Title: DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection
Article Snippet: The immunoprecipitated nucleic acid was eluted from the beads at 37°C in 100 mM Tris-HCl, pH 7.8, 10 mM EDTA, 1% SDS for 15 min, and the protein-nucleic acid cross-linking was reversed by overnight incubation at 65°C with 5 M NaCl. .. For the nuclease treatments, after the nucleic acids were released from the protein cross-link, they were ethanol precipitated and treated at 37°C with 50 U RNase A (Thermo) for 20 min in the presence of 300 mM NaCl, 4 U DNase I (Roche) with the reaction buffer provided with the enzyme for 20 min, or 3 U of RNase H (Thermo) for 20 min in the reaction buffer provided with the enzyme.

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: Cell pellets containing approximately one million crosslinked cells were resuspended in cold lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.2 % NP-40) and incubated on ice for 10 min. .. Nuclei were pelleted at 2500g for 60 s, resuspended in 100 μL of 0.5× DNase I digestion buffer [0.5× DNase I digestion buffer (Thermo), 0.5 mM MnCl2 ] containing 0.2 % SDS, and incubated at 37 °C for 30 min. An equal volume of 0.5× DNase I digestion buffer containing 2 % Triton X-100 and 4 U RNase A (Thermo) was added and incubation at 37 °C was continued for 10 min. Then, 1.5 U DNase I (Thermo) was added and digestion carried out at room temperature for 4 min. DNase I digestion was stopped by adding 40 μL of 6× Stop Solution (125 mM EDTA, 2.5 % SDS), followed by centrifugation at 2500g for 60 s. Nuclei were resuspended in 150 μL nuclease-free H2 O (Ambion), and purified with two volumes (300 μL) of AMPure XP SPRI magnetic beads (Beckman Coulter). .. The resulting mixture was well mixed, incubated at room temperature for 5 min, collected via DynaMag-Spin magnet (Invitrogen), washed twice with 80 % ethanol, and air dried for 2 min.

Article Title: The Ability of Lytic Staphylococcal Podovirus vB_SauP_phiAGO1.3 to Coexist in Equilibrium With Its Host Facilitates the Selection of Host Mutants of Attenuated Virulence but Does Not Preclude the Phage Antistaphylococcal Activity in a Nematode Infection Model
Article Snippet: Only confirmed monoclonal lysates were used for phiAGO1.3 propagation on a larger scale in 80wphwpl cells. .. Cell lysate containing phages (50 ml, 1011 PFU/ml) was filtered through 0.22-μm pore size membrane (MILLEX® GS) and treated with RNase A (PureLink RNase, Invitrogen) and DNase (Turbo DNase, Ambion) (final concentration 20 μg/ml of each) at 37°C for 2 h. Next, the lysate was supplemented with polyethylene glycol (PEG6000) and NaCl to final concentrations of 10% and 1 M, respectively, and incubated on ice for 24 h. Samples were centrifuged (10,000 × g , 30 min, 4°C) and the pellet was resuspended in phosphate buffered saline (PBS: 10 mM Na2 HPO4 , 137 mM NaCl, 2.7 mM KCl, 2 mM KH2 PO4 at pH 7.4). .. Overnight cultures of various S. aureus strains in LB medium (0.2 ml) were supplemented with CaCl2 and MgSO4 to the final concentration of 2.5 mM each, mixed with 1 ml LB and 8 ml molten LCA (55°C), overlaid on LB agar medium in Petri dishes, and left to solidify.

Article Title: Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing
Article Snippet: Fabrication of PVA/hFDM membrane To prepare a human lung fibroblast-derived matrix (hFDM), human lung fibroblasts (WI-38, CCL-75; ATCC) were seeded (2×104 /cm2 ) in 24-well plate and cultured for 7 days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin under normal culture condition (5% CO2 , 37 °C). .. After several washings with phosphate buffered saline (PBS), cells were subject to decellularization using 0.25% Triton-X 100 and 50 mM NH4 OH (221228, Sigma), followed by incubation with 50 U/mL DNase I (18047-019, Invitrogen) and 2.5 µL/mL RNase A (12091-039, Invitrogen) at 37 °C for 2 h. Once the decellularized hFDM was rinsed with PBS several times, 7% (w/v) aqueous polyvinyl alcohol (PVA) (MW 146,000-186,000; 363065, Sigma) was added onto the hFDM. .. Crosslinking of PVA hydrogel was carried out via storage at -20 °C for 24 h and subsequent thawing at room temperature for 1 h. PVA hydrogel coupled with hFDM was then carefully peeled off from the tissue culture plate using forceps and transferred to a new plate.

Article Title: Vascular Morphogenesis of Human Umbilical Vein Endothelial Cells on Cell-Derived Macromolecular Matrix Microenvironment
Article Snippet: The confluent cells were then treated briefly with a detergent solution containing 0.25% Triton X-100 and 10 mM NH4 OH (Sigma-Aldrich). .. After the samples were washed with phosphate-buffered saline (PBS), both 50 U/mL DNase I and 2.5 μL/mL RNase A (Invitrogen) in PBS were added and incubated at 37°C for 2 h, and the decellularized samples were then gently washed twice with PBS. .. Preosteoblast-derived matrix (PDM), fibroblast-derived matrix (FDM), and chondrocyte-derived matrix (CHDM) were used immediately or were stored at −70°C in 0.1 M glycine solution prior to use.

Article Title: HIV-1 remodels the nuclear pore complex
Article Snippet: In brief, equal numbers of mock- and peak-infected T cells were collected, washed with PBS, incubated in hypotonic lysis buffer (10 mM Hepes, pH 7.4, 1.5 mM MgCl2 , and 10 mM KCl) for 10 min, Dounce homogenized (40-ml capacity Wheaton type; VWR), and resuspended in 2.2 M SHKM (2.2 M sucrose, 50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ). .. Pellets were resuspended in 0.25 M SHKM (50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ), treated with RNase A and DNase I (Invitrogen) for 15 min, and resuspended in 10% SHM (0.3 M sucrose, 10 mM Hepes, pH 7.4, and 2 mM MgCl2 ) for centrifugation at 5,000 rpm for 10 min at 4°C.

Mass Spectrometry:

Article Title: HIV-1 remodels the nuclear pore complex
Article Snippet: Pellets were resuspended in 0.25 M SHKM (50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ), treated with RNase A and DNase I (Invitrogen) for 15 min, and resuspended in 10% SHM (0.3 M sucrose, 10 mM Hepes, pH 7.4, and 2 mM MgCl2 ) for centrifugation at 5,000 rpm for 10 min at 4°C. .. All solutions had 2 mM of fresh DTT and protease inhibitor (Roche) added to them.

Article Title: The Core of Chloroplast Nucleoids Contains Architectural SWIB Domain Proteins
Article Snippet: Paragraph title: Two-Dimensional Separation of Proteins and Mass Spectrometry ... Prior to , protein fractions were treated with DNase I and RNase A (MBI Fermentas) and precipitated with chloroform/methanol following the protocol of Wessel and Flügge (1984).

Release Assay:

Article Title: A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA
Article Snippet: Paragraph title: Oligonucleotide release assay ... After 20 min, Heparin (20 IE/ml) or Hirudin (20 µg/ml) were added in the same concentration as used for stimulation (see above) for 40 min, followed by DNase I (Fermentas, St. Leon Rot, Germany) or RNase A (Fermentas, St. Leon Rot, Germany) digestion at 37°C (CpG-ODN/pArg 4 U DNase I 4 h; CpG-ODN/DOTAP 3 U DNase I 4 h; 9.2 s-RNA/DOTAP 250 ng RNase A 10 min; 3p-dsRNA/Lipofectamine 500 ng RNase A 90 min).

Modification:

Article Title: Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing
Article Snippet: Fabrication of PVA/hFDM membrane To prepare a human lung fibroblast-derived matrix (hFDM), human lung fibroblasts (WI-38, CCL-75; ATCC) were seeded (2×104 /cm2 ) in 24-well plate and cultured for 7 days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin under normal culture condition (5% CO2 , 37 °C). .. After several washings with phosphate buffered saline (PBS), cells were subject to decellularization using 0.25% Triton-X 100 and 50 mM NH4 OH (221228, Sigma), followed by incubation with 50 U/mL DNase I (18047-019, Invitrogen) and 2.5 µL/mL RNase A (12091-039, Invitrogen) at 37 °C for 2 h. Once the decellularized hFDM was rinsed with PBS several times, 7% (w/v) aqueous polyvinyl alcohol (PVA) (MW 146,000-186,000; 363065, Sigma) was added onto the hFDM.

Article Title: Vascular Morphogenesis of Human Umbilical Vein Endothelial Cells on Cell-Derived Macromolecular Matrix Microenvironment
Article Snippet: NIH3T3 mouse fibroblasts, human chondrocytes, and MC3T3-E1 mouse preosteoblasts (ATCC) were cultured in Dulbecco's modified Eagle's medium or minimal essential alpha medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). .. After the samples were washed with phosphate-buffered saline (PBS), both 50 U/mL DNase I and 2.5 μL/mL RNase A (Invitrogen) in PBS were added and incubated at 37°C for 2 h, and the decellularized samples were then gently washed twice with PBS.

Transformation Assay:

Article Title: Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications
Article Snippet: Transformed cells growing in LB medium were induced at A600nm = 0.6 with 0.6 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 24 h at 18°C. .. Cells were harvested and lyzed in lysis buffer (50 mM TRIS·HCl, pH = 8.0, 300 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1×cOmplete ULTRA™ EDTA free protease inhibitor cocktail tablet (Roche), 0.1 mg/ml lysozyme, 0.1 mg/ml DNase (Sigma, St. Louis, MO, USA) and 0.01 mg/ml RNAse A (Invitrogen, Carlsbad, CA, USA)) assisted with sonication.

Concentration Assay:

Article Title: A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA
Article Snippet: CpG 2006, 9.2 s and 3p-dsRNA were incubated with corresponding delivery agent as described above. .. After 20 min, Heparin (20 IE/ml) or Hirudin (20 µg/ml) were added in the same concentration as used for stimulation (see above) for 40 min, followed by DNase I (Fermentas, St. Leon Rot, Germany) or RNase A (Fermentas, St. Leon Rot, Germany) digestion at 37°C (CpG-ODN/pArg 4 U DNase I 4 h; CpG-ODN/DOTAP 3 U DNase I 4 h; 9.2 s-RNA/DOTAP 250 ng RNase A 10 min; 3p-dsRNA/Lipofectamine 500 ng RNase A 90 min). .. RNase A was inactivated by adding 280 U RiboLock (Fermentas, St. Leon Rot, Germany) for 15 min and DNase I by heating to 65°C for 10 min. After addition of Heparin to displace all nucleic acids from delivery agent, the mixture was loaded on a native polyacrylamide gel (15%).

Article Title: Characterization of Bacteriophage vB-EcoS-95, Isolated From Urban Sewage and Revealing Extremely Rapid Lytic Development
Article Snippet: The phage lysate was treated with DNase I (1 U/μl; Thermo Fisher Scientific) and RNase A (5 ug/μl; Thermo Fisher Scientific) to degrade bacterial nucleic acids. .. Then, DNase I and RNase A were inactivated by heating to 95°C and the genomic DNA of phage vB_EcoS-95 was isolated with a MasterPure™ Complete DNA and RNA Purification Kit (Epicenter).

Article Title: The Ability of Lytic Staphylococcal Podovirus vB_SauP_phiAGO1.3 to Coexist in Equilibrium With Its Host Facilitates the Selection of Host Mutants of Attenuated Virulence but Does Not Preclude the Phage Antistaphylococcal Activity in a Nematode Infection Model
Article Snippet: Only confirmed monoclonal lysates were used for phiAGO1.3 propagation on a larger scale in 80wphwpl cells. .. Cell lysate containing phages (50 ml, 1011 PFU/ml) was filtered through 0.22-μm pore size membrane (MILLEX® GS) and treated with RNase A (PureLink RNase, Invitrogen) and DNase (Turbo DNase, Ambion) (final concentration 20 μg/ml of each) at 37°C for 2 h. Next, the lysate was supplemented with polyethylene glycol (PEG6000) and NaCl to final concentrations of 10% and 1 M, respectively, and incubated on ice for 24 h. Samples were centrifuged (10,000 × g , 30 min, 4°C) and the pellet was resuspended in phosphate buffered saline (PBS: 10 mM Na2 HPO4 , 137 mM NaCl, 2.7 mM KCl, 2 mM KH2 PO4 at pH 7.4). .. Overnight cultures of various S. aureus strains in LB medium (0.2 ml) were supplemented with CaCl2 and MgSO4 to the final concentration of 2.5 mM each, mixed with 1 ml LB and 8 ml molten LCA (55°C), overlaid on LB agar medium in Petri dishes, and left to solidify.

Protease Inhibitor:

Article Title: Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System
Article Snippet: This protocol will facilitate detailed studies on capsid assembly and host regulation under physiological conditions and identification of novel antiviral agents targeting HBc. .. Pipette tips (Denville Scientific, catalog numbers: P1096-FR, P1121, P1122, P1126) 1.5 ml microcentrifuge tube (Denville Scientific, catalog number: C2170) Gloves and lab coat (Denville Scientific, catalog number: G4162; Medline Industries, catalog number: 83044QHW) Proteinase K, RNA grade (Thermo Fisher Scientific, Invitrogen™, catalog number: 25530049) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509-1KG) Phenol solution (Sigma-Aldrich, catalog number: P4557) Chloroform (Fisher Scientific, catalog number: BP1145-1) Sodium acetate (Sigma-Aldrich, catalog number: S2889-1KG) Ethyl alcohol (EtOH) (AmericanBio, catalog number: AB00515-00100) UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, Invitrogen™, catalog number: 10977015) Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 15510027) TNT® Coupled Rabbit Reticulocyte Lysate (RRL) (Promega, catalog number: L4610) EasyTag™ L-[35 S]-Methionine (PerkinElmer, catalog number: NEG709A001MC) RNasin® Plus Ribonuclease Inhibitor (Promega, catalog number: N2611) RNaseZap™ RNase Decontamination Solution (Thermo Fisher Scientific, Invitrogen™, catalog number: AM9780) RNase A, DNase and protease-free (Thermo Fisher Scientific, Thermo Scientific™, catalog number: EN0531) NEBuffer 3 (New England Biolabs, catalog number: B7003S) Alkaline Phosphatase, Calf Intestinal (CIAP) (New England Biolabs, catalog number: M0290S) Tris Base (Fisher Scientific, catalog number: BP152-10) Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: E5134) Sodium fluoride (Sigma-Aldrich, catalog number: S7920) Sodium pyrophosphate tetrabasic decahydrate (Sigma-Aldrich, catalog number: S6422) β-Glycerophosphate (Sigma-Aldrich, catalog number: G6251) Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508) cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 04693132001) TE buffer (see Recipes) 10× phosphatase inhibitors (PPI) (see Recipes) 25× protease inhibitor (see Recipes) .. Pipettes (Gilson, P1000, P200, P20, P2) Fume hood ( e.g. , Protector Xstream Laboratory Hood, Labconco) 30 °C/37 °C Oven (SciGene, Robbins Scientific, model: Model 400) Microcentrifuge (Fisher Scientific, Fisherbrand™, model: accuSpin™ Micro 17) Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific™, model: NanoDrop™1000)

Article Title: Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications
Article Snippet: Transformed cells growing in LB medium were induced at A600nm = 0.6 with 0.6 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 24 h at 18°C. .. Cells were harvested and lyzed in lysis buffer (50 mM TRIS·HCl, pH = 8.0, 300 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1×cOmplete ULTRA™ EDTA free protease inhibitor cocktail tablet (Roche), 0.1 mg/ml lysozyme, 0.1 mg/ml DNase (Sigma, St. Louis, MO, USA) and 0.01 mg/ml RNAse A (Invitrogen, Carlsbad, CA, USA)) assisted with sonication. .. Cell debris was pelleted by centrifugation at 20 000 × g for 30 min. Supernatant was applied onto a Ni-NTA column and washed with a set of washing buffers: low salt buffer (50 mM HEPES, pH = 7.5, 30 mM KCl, 5 mM β-mercaptoethanol), high salt buffer (50 mM HEPES, pH = 7.5, 300 mM KCl, 5 mM β-mercaptoethanol) and very high salt buffer (50 mM HEPES, pH = 7.5, 500 mM NaCl, 40 mM Imidazole, 5 mM β-mercaptoethanol).

Article Title: HIV-1 remodels the nuclear pore complex
Article Snippet: Pellets were resuspended in 0.25 M SHKM (50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ), treated with RNase A and DNase I (Invitrogen) for 15 min, and resuspended in 10% SHM (0.3 M sucrose, 10 mM Hepes, pH 7.4, and 2 mM MgCl2 ) for centrifugation at 5,000 rpm for 10 min at 4°C. .. Pellets were resuspended in 0.25 M SHKM (50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ), treated with RNase A and DNase I (Invitrogen) for 15 min, and resuspended in 10% SHM (0.3 M sucrose, 10 mM Hepes, pH 7.4, and 2 mM MgCl2 ) for centrifugation at 5,000 rpm for 10 min at 4°C.

Transferring:

Article Title: Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System
Article Snippet: This protocol will facilitate detailed studies on capsid assembly and host regulation under physiological conditions and identification of novel antiviral agents targeting HBc. .. Pipette tips (Denville Scientific, catalog numbers: P1096-FR, P1121, P1122, P1126) 1.5 ml microcentrifuge tube (Denville Scientific, catalog number: C2170) Gloves and lab coat (Denville Scientific, catalog number: G4162; Medline Industries, catalog number: 83044QHW) Proteinase K, RNA grade (Thermo Fisher Scientific, Invitrogen™, catalog number: 25530049) Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509-1KG) Phenol solution (Sigma-Aldrich, catalog number: P4557) Chloroform (Fisher Scientific, catalog number: BP1145-1) Sodium acetate (Sigma-Aldrich, catalog number: S2889-1KG) Ethyl alcohol (EtOH) (AmericanBio, catalog number: AB00515-00100) UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, Invitrogen™, catalog number: 10977015) Agarose (Thermo Fisher Scientific, Invitrogen™, catalog number: 15510027) TNT® Coupled Rabbit Reticulocyte Lysate (RRL) (Promega, catalog number: L4610) EasyTag™ L-[35 S]-Methionine (PerkinElmer, catalog number: NEG709A001MC) RNasin® Plus Ribonuclease Inhibitor (Promega, catalog number: N2611) RNaseZap™ RNase Decontamination Solution (Thermo Fisher Scientific, Invitrogen™, catalog number: AM9780) RNase A, DNase and protease-free (Thermo Fisher Scientific, Thermo Scientific™, catalog number: EN0531) NEBuffer 3 (New England Biolabs, catalog number: B7003S) Alkaline Phosphatase, Calf Intestinal (CIAP) (New England Biolabs, catalog number: M0290S) Tris Base (Fisher Scientific, catalog number: BP152-10) Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: E5134) Sodium fluoride (Sigma-Aldrich, catalog number: S7920) Sodium pyrophosphate tetrabasic decahydrate (Sigma-Aldrich, catalog number: S6422) β-Glycerophosphate (Sigma-Aldrich, catalog number: G6251) Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508) cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, catalog number: 04693132001) TE buffer (see Recipes) 10× phosphatase inhibitors (PPI) (see Recipes) 25× protease inhibitor (see Recipes) .. Pipettes (Gilson, P1000, P200, P20, P2) Fume hood ( e.g. , Protector Xstream Laboratory Hood, Labconco) 30 °C/37 °C Oven (SciGene, Robbins Scientific, model: Model 400) Microcentrifuge (Fisher Scientific, Fisherbrand™, model: accuSpin™ Micro 17) Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific™, model: NanoDrop™1000)

Cell Culture:

Article Title: Induction of chondrogenesis of human placenta-derived mesenchymal stem cells via heparin-grafted human fibroblast derived matrix
Article Snippet: WI-38 human lung fibroblasts (ATCC, CCL-75) were cultured at the cell density of 2 × 104 cells/cm2 on the tissue culture dish (100 mm diameter) for 7 days in the DMEM supplemented with 10% FBS and 1% P/S. .. Once confluent, cell-loaded culture dish was washed twice with PBS, incubated briefly in a detergent solution containing 0.15% Triton X-100 (AMRESCO, Inc., Dallas, USA) and 10 mM NH4 OH (Sigma; St. Louis, MO, USA) at 37 °C, and then treated with 50 U/mL DNase I and 50 μg/mL RNase A (Invitrogen) for 1 h. After the decellularization process, ECMs were collected into centrifuge tubes and stored at 4 °C for future usage.

Article Title: Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing
Article Snippet: Fabrication of PVA/hFDM membrane To prepare a human lung fibroblast-derived matrix (hFDM), human lung fibroblasts (WI-38, CCL-75; ATCC) were seeded (2×104 /cm2 ) in 24-well plate and cultured for 7 days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin under normal culture condition (5% CO2 , 37 °C). .. After several washings with phosphate buffered saline (PBS), cells were subject to decellularization using 0.25% Triton-X 100 and 50 mM NH4 OH (221228, Sigma), followed by incubation with 50 U/mL DNase I (18047-019, Invitrogen) and 2.5 µL/mL RNase A (12091-039, Invitrogen) at 37 °C for 2 h. Once the decellularized hFDM was rinsed with PBS several times, 7% (w/v) aqueous polyvinyl alcohol (PVA) (MW 146,000-186,000; 363065, Sigma) was added onto the hFDM.

Article Title: Vascular Morphogenesis of Human Umbilical Vein Endothelial Cells on Cell-Derived Macromolecular Matrix Microenvironment
Article Snippet: NIH3T3 mouse fibroblasts, human chondrocytes, and MC3T3-E1 mouse preosteoblasts (ATCC) were cultured in Dulbecco's modified Eagle's medium or minimal essential alpha medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). .. After the samples were washed with phosphate-buffered saline (PBS), both 50 U/mL DNase I and 2.5 μL/mL RNase A (Invitrogen) in PBS were added and incubated at 37°C for 2 h, and the decellularized samples were then gently washed twice with PBS.

Light Microscopy:

Article Title: Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing
Article Snippet: After several washings with phosphate buffered saline (PBS), cells were subject to decellularization using 0.25% Triton-X 100 and 50 mM NH4 OH (221228, Sigma), followed by incubation with 50 U/mL DNase I (18047-019, Invitrogen) and 2.5 µL/mL RNase A (12091-039, Invitrogen) at 37 °C for 2 h. Once the decellularized hFDM was rinsed with PBS several times, 7% (w/v) aqueous polyvinyl alcohol (PVA) (MW 146,000-186,000; 363065, Sigma) was added onto the hFDM. .. After several washings with phosphate buffered saline (PBS), cells were subject to decellularization using 0.25% Triton-X 100 and 50 mM NH4 OH (221228, Sigma), followed by incubation with 50 U/mL DNase I (18047-019, Invitrogen) and 2.5 µL/mL RNase A (12091-039, Invitrogen) at 37 °C for 2 h. Once the decellularized hFDM was rinsed with PBS several times, 7% (w/v) aqueous polyvinyl alcohol (PVA) (MW 146,000-186,000; 363065, Sigma) was added onto the hFDM.

Generated:

Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity
Article Snippet: Homologous RNA without 5′ppp moiety was purchased from IDT (Integrated DNA Technologies Inc, Iowa, USA); dephosphorylation of the 5′pppRNA using CIAP (Invitrogen, NY, USA) generated identical results (data not shown). .. RNA was analysed on a denaturing 17% polyacrylamide, 7 M urea gel following digestion with 50 ng/ul of RNase A (Ambion, CA, USA) or 100 mU/ul of DNase I (Ambion, CA, USA) for 30 min.

Article Title: Identification of a Divergent Lineage Porcine Pestivirus in Nursing Piglets with Congenital Tremors and Reproduction of Disease following Experimental Inoculation
Article Snippet: Serum or tissue homogenates were re-suspended in Hanks balanced salt solution (Corning-Cellgro) and enriched for viral particle protected nucleic acids by digestion with a combination of nucleases: RNase A (Invitrogen), Baseline Zero DNase (Epicentre), and Turbo DNase (Invitrogen) as described previously [ ]. .. Post-extraction, nucleic acids were further treated with Turbo DNase to remove host or potential viral DNA, thus further enriching for viral RNA.

Reverse Transcription Polymerase Chain Reaction:

Article Title: DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection
Article Snippet: The eluted nucleic acid was purified using the DNeasy kit (Qiagen) and analyzed with RT-PCR strong-stop primers (primers PR and PU5 in ) or 3′ LTR primers (primers P3′R -P3′L in ) ( ). .. For the nuclease treatments, after the nucleic acids were released from the protein cross-link, they were ethanol precipitated and treated at 37°C with 50 U RNase A (Thermo) for 20 min in the presence of 300 mM NaCl, 4 U DNase I (Roche) with the reaction buffer provided with the enzyme for 20 min, or 3 U of RNase H (Thermo) for 20 min in the reaction buffer provided with the enzyme.

Sonication:

Article Title: Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications
Article Snippet: Transformed cells growing in LB medium were induced at A600nm = 0.6 with 0.6 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 24 h at 18°C. .. Cells were harvested and lyzed in lysis buffer (50 mM TRIS·HCl, pH = 8.0, 300 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1×cOmplete ULTRA™ EDTA free protease inhibitor cocktail tablet (Roche), 0.1 mg/ml lysozyme, 0.1 mg/ml DNase (Sigma, St. Louis, MO, USA) and 0.01 mg/ml RNAse A (Invitrogen, Carlsbad, CA, USA)) assisted with sonication. .. Cell debris was pelleted by centrifugation at 20 000 × g for 30 min. Supernatant was applied onto a Ni-NTA column and washed with a set of washing buffers: low salt buffer (50 mM HEPES, pH = 7.5, 30 mM KCl, 5 mM β-mercaptoethanol), high salt buffer (50 mM HEPES, pH = 7.5, 300 mM KCl, 5 mM β-mercaptoethanol) and very high salt buffer (50 mM HEPES, pH = 7.5, 500 mM NaCl, 40 mM Imidazole, 5 mM β-mercaptoethanol).

Phage Purification:

Article Title: The Ability of Lytic Staphylococcal Podovirus vB_SauP_phiAGO1.3 to Coexist in Equilibrium With Its Host Facilitates the Selection of Host Mutants of Attenuated Virulence but Does Not Preclude the Phage Antistaphylococcal Activity in a Nematode Infection Model
Article Snippet: Paragraph title: Phage Purification ... Cell lysate containing phages (50 ml, 1011 PFU/ml) was filtered through 0.22-μm pore size membrane (MILLEX® GS) and treated with RNase A (PureLink RNase, Invitrogen) and DNase (Turbo DNase, Ambion) (final concentration 20 μg/ml of each) at 37°C for 2 h. Next, the lysate was supplemented with polyethylene glycol (PEG6000) and NaCl to final concentrations of 10% and 1 M, respectively, and incubated on ice for 24 h. Samples were centrifuged (10,000 × g , 30 min, 4°C) and the pellet was resuspended in phosphate buffered saline (PBS: 10 mM Na2 HPO4 , 137 mM NaCl, 2.7 mM KCl, 2 mM KH2 PO4 at pH 7.4).

Recombinant:

Article Title: Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications
Article Snippet: Paragraph title: Recombinant protein production ... Cells were harvested and lyzed in lysis buffer (50 mM TRIS·HCl, pH = 8.0, 300 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1×cOmplete ULTRA™ EDTA free protease inhibitor cocktail tablet (Roche), 0.1 mg/ml lysozyme, 0.1 mg/ml DNase (Sigma, St. Louis, MO, USA) and 0.01 mg/ml RNAse A (Invitrogen, Carlsbad, CA, USA)) assisted with sonication.

Cellular Antioxidant Activity Assay:

Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity
Article Snippet: The template consisted of two complementary viral sequences containing T7 promoter that were annealed at 95°C for 5 minutes and cooled down gradually over night ( 5′-GAC GAA GAC AAA CAA ACC ATT ATT ATC ATT AAA ATT TTA TTT TTT ATC TGG TTT TGT GGT CTT CGT CTA TAG TGA GTC GTA TTA ATT TC-3′ ). .. RNA was analysed on a denaturing 17% polyacrylamide, 7 M urea gel following digestion with 50 ng/ul of RNase A (Ambion, CA, USA) or 100 mU/ul of DNase I (Ambion, CA, USA) for 30 min.

DNA Extraction:

Article Title: Characterization of Bacteriophage vB-EcoS-95, Isolated From Urban Sewage and Revealing Extremely Rapid Lytic Development
Article Snippet: Paragraph title: Phage DNA Isolation ... The phage lysate was treated with DNase I (1 U/μl; Thermo Fisher Scientific) and RNase A (5 ug/μl; Thermo Fisher Scientific) to degrade bacterial nucleic acids.

Magnetic Beads:

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: Cell pellets containing approximately one million crosslinked cells were resuspended in cold lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.2 % NP-40) and incubated on ice for 10 min. .. Nuclei were pelleted at 2500g for 60 s, resuspended in 100 μL of 0.5× DNase I digestion buffer [0.5× DNase I digestion buffer (Thermo), 0.5 mM MnCl2 ] containing 0.2 % SDS, and incubated at 37 °C for 30 min. An equal volume of 0.5× DNase I digestion buffer containing 2 % Triton X-100 and 4 U RNase A (Thermo) was added and incubation at 37 °C was continued for 10 min. Then, 1.5 U DNase I (Thermo) was added and digestion carried out at room temperature for 4 min. DNase I digestion was stopped by adding 40 μL of 6× Stop Solution (125 mM EDTA, 2.5 % SDS), followed by centrifugation at 2500g for 60 s. Nuclei were resuspended in 150 μL nuclease-free H2 O (Ambion), and purified with two volumes (300 μL) of AMPure XP SPRI magnetic beads (Beckman Coulter). .. The resulting mixture was well mixed, incubated at room temperature for 5 min, collected via DynaMag-Spin magnet (Invitrogen), washed twice with 80 % ethanol, and air dried for 2 min.

Isolation:

Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity
Article Snippet: 5′pppRNA was purified and isolated using the Qiagen miRNA Mini Kit (MD, USA). .. RNA was analysed on a denaturing 17% polyacrylamide, 7 M urea gel following digestion with 50 ng/ul of RNase A (Ambion, CA, USA) or 100 mU/ul of DNase I (Ambion, CA, USA) for 30 min.

Article Title: Characterization of Bacteriophage vB-EcoS-95, Isolated From Urban Sewage and Revealing Extremely Rapid Lytic Development
Article Snippet: The phage lysate was treated with DNase I (1 U/μl; Thermo Fisher Scientific) and RNase A (5 ug/μl; Thermo Fisher Scientific) to degrade bacterial nucleic acids. .. To digest the exogenous DNA and RNA, the mixture was incubated for 30 min at 37°C.

Article Title: The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules
Article Snippet: Paragraph title: 2.8. Genome Isolation and Sequencing ... The purified nucleic acid was treated with RQ1 DNase (Promega; Madison, WI, USA), RNase A (Fermentas, Thermo Fisher), Exonuclease III (Fermentas), Bal31 nuclease (New England Biolabs, Ipswich, MA, USA), as well as with restriction enzymes: AseI, SalI, HincII, MseI, SspI, SmaI, MscI, NruI (New England Biolabs), NotI (Fermentas), and HindIII (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

Article Title: HIV-1 remodels the nuclear pore complex
Article Snippet: Paragraph title: NE isolation ... Pellets were resuspended in 0.25 M SHKM (50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ), treated with RNase A and DNase I (Invitrogen) for 15 min, and resuspended in 10% SHM (0.3 M sucrose, 10 mM Hepes, pH 7.4, and 2 mM MgCl2 ) for centrifugation at 5,000 rpm for 10 min at 4°C.

Labeling:

Article Title: Hepatitis B and Hepatitis C Infection Biomarkers and TP53 Mutations in Hepatocellular Carcinomas from Colombia
Article Snippet: The liver tissue sections were labeled using the antibody highly specific for AFB1-Fapy adducts 6A10, developed and characterized by Hsieh et al. [ ]. .. The liver sections were then treated with RNase A (100 μ L/mL, Fermentas, RNase A , DNase , and Protease-free ), with Proteinase K for 10 min at 37°C (10 μ L/mL, Gentra Puregene) and with NaOH 50 mM in 40% ethanol for DNA denaturation.

Purification:

Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity
Article Snippet: 5′pppRNA was purified and isolated using the Qiagen miRNA Mini Kit (MD, USA). .. RNA was analysed on a denaturing 17% polyacrylamide, 7 M urea gel following digestion with 50 ng/ul of RNase A (Ambion, CA, USA) or 100 mU/ul of DNase I (Ambion, CA, USA) for 30 min.

Article Title: DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection
Article Snippet: The eluted nucleic acid was purified using the DNeasy kit (Qiagen) and analyzed with RT-PCR strong-stop primers (primers PR and PU5 in ) or 3′ LTR primers (primers P3′R -P3′L in ) ( ). .. For the nuclease treatments, after the nucleic acids were released from the protein cross-link, they were ethanol precipitated and treated at 37°C with 50 U RNase A (Thermo) for 20 min in the presence of 300 mM NaCl, 4 U DNase I (Roche) with the reaction buffer provided with the enzyme for 20 min, or 3 U of RNase H (Thermo) for 20 min in the reaction buffer provided with the enzyme.

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: Cell pellets containing approximately one million crosslinked cells were resuspended in cold lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.2 % NP-40) and incubated on ice for 10 min. .. Nuclei were pelleted at 2500g for 60 s, resuspended in 100 μL of 0.5× DNase I digestion buffer [0.5× DNase I digestion buffer (Thermo), 0.5 mM MnCl2 ] containing 0.2 % SDS, and incubated at 37 °C for 30 min. An equal volume of 0.5× DNase I digestion buffer containing 2 % Triton X-100 and 4 U RNase A (Thermo) was added and incubation at 37 °C was continued for 10 min. Then, 1.5 U DNase I (Thermo) was added and digestion carried out at room temperature for 4 min. DNase I digestion was stopped by adding 40 μL of 6× Stop Solution (125 mM EDTA, 2.5 % SDS), followed by centrifugation at 2500g for 60 s. Nuclei were resuspended in 150 μL nuclease-free H2 O (Ambion), and purified with two volumes (300 μL) of AMPure XP SPRI magnetic beads (Beckman Coulter). .. The resulting mixture was well mixed, incubated at room temperature for 5 min, collected via DynaMag-Spin magnet (Invitrogen), washed twice with 80 % ethanol, and air dried for 2 min.

Article Title: Characterization of Bacteriophage vB-EcoS-95, Isolated From Urban Sewage and Revealing Extremely Rapid Lytic Development
Article Snippet: The phage lysate was treated with DNase I (1 U/μl; Thermo Fisher Scientific) and RNase A (5 ug/μl; Thermo Fisher Scientific) to degrade bacterial nucleic acids. .. To digest the exogenous DNA and RNA, the mixture was incubated for 30 min at 37°C.

Article Title: The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules
Article Snippet: Phenol-ether extraction of the nucleic acid was followed by precipitation by NaCl and ethanol. .. The purified nucleic acid was treated with RQ1 DNase (Promega; Madison, WI, USA), RNase A (Fermentas, Thermo Fisher), Exonuclease III (Fermentas), Bal31 nuclease (New England Biolabs, Ipswich, MA, USA), as well as with restriction enzymes: AseI, SalI, HincII, MseI, SspI, SmaI, MscI, NruI (New England Biolabs), NotI (Fermentas), and HindIII (Roche, Basel, Switzerland) according to the manufacturer’s instructions. .. Untreated virus DNA, DNA treated with Sulfolobus polymerase IV and T4 ligase, and DNA treated with Klenow fragment and T4 ligase were pooled together and sequenced using Illumina MiSeq (Illumina, San Diego, CA, USA).

Sequencing:

Article Title: Identification of a Divergent Lineage Porcine Pestivirus in Nursing Piglets with Congenital Tremors and Reproduction of Disease following Experimental Inoculation
Article Snippet: With the exception of the lung tissue from sample ID 20120705, all samples tested exhibited at least partial pestivirus genomic sequence. .. Serum or tissue homogenates were re-suspended in Hanks balanced salt solution (Corning-Cellgro) and enriched for viral particle protected nucleic acids by digestion with a combination of nucleases: RNase A (Invitrogen), Baseline Zero DNase (Epicentre), and Turbo DNase (Invitrogen) as described previously [ ].

Article Title: The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules
Article Snippet: Paragraph title: 2.8. Genome Isolation and Sequencing ... The purified nucleic acid was treated with RQ1 DNase (Promega; Madison, WI, USA), RNase A (Fermentas, Thermo Fisher), Exonuclease III (Fermentas), Bal31 nuclease (New England Biolabs, Ipswich, MA, USA), as well as with restriction enzymes: AseI, SalI, HincII, MseI, SspI, SmaI, MscI, NruI (New England Biolabs), NotI (Fermentas), and HindIII (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

Article Title: The Core of Chloroplast Nucleoids Contains Architectural SWIB Domain Proteins
Article Snippet: Prior to , protein fractions were treated with DNase I and RNase A (MBI Fermentas) and precipitated with chloroform/methanol following the protocol of Wessel and Flügge (1984). .. Separated proteins were stained with colloidal Coomassie Brilliant Blue (Gel Code Blue; Thermo Fisher Scientific).

De-Phosphorylation Assay:

Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity
Article Snippet: Homologous RNA without 5′ppp moiety was purchased from IDT (Integrated DNA Technologies Inc, Iowa, USA); dephosphorylation of the 5′pppRNA using CIAP (Invitrogen, NY, USA) generated identical results (data not shown). .. RNA was analysed on a denaturing 17% polyacrylamide, 7 M urea gel following digestion with 50 ng/ul of RNase A (Ambion, CA, USA) or 100 mU/ul of DNase I (Ambion, CA, USA) for 30 min.

Staining:

Article Title: A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA
Article Snippet: After 20 min, Heparin (20 IE/ml) or Hirudin (20 µg/ml) were added in the same concentration as used for stimulation (see above) for 40 min, followed by DNase I (Fermentas, St. Leon Rot, Germany) or RNase A (Fermentas, St. Leon Rot, Germany) digestion at 37°C (CpG-ODN/pArg 4 U DNase I 4 h; CpG-ODN/DOTAP 3 U DNase I 4 h; 9.2 s-RNA/DOTAP 250 ng RNase A 10 min; 3p-dsRNA/Lipofectamine 500 ng RNase A 90 min). .. After 20 min, Heparin (20 IE/ml) or Hirudin (20 µg/ml) were added in the same concentration as used for stimulation (see above) for 40 min, followed by DNase I (Fermentas, St. Leon Rot, Germany) or RNase A (Fermentas, St. Leon Rot, Germany) digestion at 37°C (CpG-ODN/pArg 4 U DNase I 4 h; CpG-ODN/DOTAP 3 U DNase I 4 h; 9.2 s-RNA/DOTAP 250 ng RNase A 10 min; 3p-dsRNA/Lipofectamine 500 ng RNase A 90 min).

Article Title: The Core of Chloroplast Nucleoids Contains Architectural SWIB Domain Proteins
Article Snippet: Prior to , protein fractions were treated with DNase I and RNase A (MBI Fermentas) and precipitated with chloroform/methanol following the protocol of Wessel and Flügge (1984). .. Prior to , protein fractions were treated with DNase I and RNase A (MBI Fermentas) and precipitated with chloroform/methanol following the protocol of Wessel and Flügge (1984).

Sample Prep:

Article Title: Viral diversity of Rhipicephalus microplus parasitizing cattle in southern Brazil
Article Snippet: Paragraph title: Sample preparation ... To remove the naked DNA and RNA, 200 μl of the resuspended pellet from each pooled sample were digested in a cocktail with 20U of Turbo DNase (Life Technologies, USA), 25U of benzonase (Sigma-Aldrich, USA), and 0.1 mg/ml of RNase A (Life Technologies, USA) at 37 °C for 2 hours in 20 μl of 10X DNase buffer (Life Technologies, USA).

Electrophoresis:

Article Title: Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines
Article Snippet: Paragraph title: Two-dimensional gel electrophoresis (2-DE) analysis ... Cell pellets of TMZ-treated and untreated samples were solubilised in 0.1 ml 40 mM Tris supplemented with 20 μg/ml RNase A (bovine pancreas source; USB Corporation), 50 U/ml DNase I (bovine pancreas source; USB Corporation), complete ethylenediamine tetraacetic acid (EDTA)-free protease inhibitors (Roche Diagnostics) and 0.5 mM phenylmethylsulphonylfluoride (PMSF) (USB Corporation), and lysed by vortexing.

Agarose Gel Electrophoresis:

Article Title: Characterization of Four Novel Bacteriophages Isolated from British Columbia for Control of Non-typhoidal Salmonella in Vitro and on Sprouting Alfalfa Seeds
Article Snippet: Prior to nucleic acid extraction, DNase I (Invitrogen, Carlsbad, CA, United States) and RNAse A (Invitrogen) were added to high titer phage lysates (109 – 1011 PFU/ml) to final concentrations of 10 and 55 μg/ml, respectively, for degradation of host nucleic acid , followed by incubation at 37°C for 30 min. Phage nucleic acid was then extracted with the PureLink Viral RNA/DNA Mini Kit (Thermo Fisher) as per the manufacturer’s instructions. .. Prior to nucleic acid extraction, DNase I (Invitrogen, Carlsbad, CA, United States) and RNAse A (Invitrogen) were added to high titer phage lysates (109 – 1011 PFU/ml) to final concentrations of 10 and 55 μg/ml, respectively, for degradation of host nucleic acid , followed by incubation at 37°C for 30 min. Phage nucleic acid was then extracted with the PureLink Viral RNA/DNA Mini Kit (Thermo Fisher) as per the manufacturer’s instructions.

In Vitro:

Article Title: Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity
Article Snippet: Paragraph title: In vitro synthesis of 5′pppRNAs ... RNA was analysed on a denaturing 17% polyacrylamide, 7 M urea gel following digestion with 50 ng/ul of RNase A (Ambion, CA, USA) or 100 mU/ul of DNase I (Ambion, CA, USA) for 30 min.

Next-Generation Sequencing:

Article Title: Identification of a Divergent Lineage Porcine Pestivirus in Nursing Piglets with Congenital Tremors and Reproduction of Disease following Experimental Inoculation
Article Snippet: Paragraph title: Next-generation sequencing ... Serum or tissue homogenates were re-suspended in Hanks balanced salt solution (Corning-Cellgro) and enriched for viral particle protected nucleic acids by digestion with a combination of nucleases: RNase A (Invitrogen), Baseline Zero DNase (Epicentre), and Turbo DNase (Invitrogen) as described previously [ ].

Immunoprecipitation:

Article Title: DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for In Vivo Control of Murine Leukemia Virus Infection
Article Snippet: The immunoprecipitated nucleic acid was eluted from the beads at 37°C in 100 mM Tris-HCl, pH 7.8, 10 mM EDTA, 1% SDS for 15 min, and the protein-nucleic acid cross-linking was reversed by overnight incubation at 65°C with 5 M NaCl. .. For the nuclease treatments, after the nucleic acids were released from the protein cross-link, they were ethanol precipitated and treated at 37°C with 50 U RNase A (Thermo) for 20 min in the presence of 300 mM NaCl, 4 U DNase I (Roche) with the reaction buffer provided with the enzyme for 20 min, or 3 U of RNase H (Thermo) for 20 min in the reaction buffer provided with the enzyme.

Two-Dimensional Gel Electrophoresis:

Article Title: Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines
Article Snippet: Paragraph title: Two-dimensional gel electrophoresis (2-DE) analysis ... Cell pellets of TMZ-treated and untreated samples were solubilised in 0.1 ml 40 mM Tris supplemented with 20 μg/ml RNase A (bovine pancreas source; USB Corporation), 50 U/ml DNase I (bovine pancreas source; USB Corporation), complete ethylenediamine tetraacetic acid (EDTA)-free protease inhibitors (Roche Diagnostics) and 0.5 mM phenylmethylsulphonylfluoride (PMSF) (USB Corporation), and lysed by vortexing.

Lysis:

Article Title: Bipartite structure of the inactive mouse X chromosome
Article Snippet: Cell pellets containing approximately one million crosslinked cells were resuspended in cold lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl, 0.2 % NP-40) and incubated on ice for 10 min. .. Nuclei were pelleted at 2500g for 60 s, resuspended in 100 μL of 0.5× DNase I digestion buffer [0.5× DNase I digestion buffer (Thermo), 0.5 mM MnCl2 ] containing 0.2 % SDS, and incubated at 37 °C for 30 min. An equal volume of 0.5× DNase I digestion buffer containing 2 % Triton X-100 and 4 U RNase A (Thermo) was added and incubation at 37 °C was continued for 10 min. Then, 1.5 U DNase I (Thermo) was added and digestion carried out at room temperature for 4 min. DNase I digestion was stopped by adding 40 μL of 6× Stop Solution (125 mM EDTA, 2.5 % SDS), followed by centrifugation at 2500g for 60 s. Nuclei were resuspended in 150 μL nuclease-free H2 O (Ambion), and purified with two volumes (300 μL) of AMPure XP SPRI magnetic beads (Beckman Coulter).

Article Title: Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications
Article Snippet: Transformed cells growing in LB medium were induced at A600nm = 0.6 with 0.6 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 24 h at 18°C. .. Cells were harvested and lyzed in lysis buffer (50 mM TRIS·HCl, pH = 8.0, 300 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 10 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 1×cOmplete ULTRA™ EDTA free protease inhibitor cocktail tablet (Roche), 0.1 mg/ml lysozyme, 0.1 mg/ml DNase (Sigma, St. Louis, MO, USA) and 0.01 mg/ml RNAse A (Invitrogen, Carlsbad, CA, USA)) assisted with sonication. .. Cell debris was pelleted by centrifugation at 20 000 × g for 30 min. Supernatant was applied onto a Ni-NTA column and washed with a set of washing buffers: low salt buffer (50 mM HEPES, pH = 7.5, 30 mM KCl, 5 mM β-mercaptoethanol), high salt buffer (50 mM HEPES, pH = 7.5, 300 mM KCl, 5 mM β-mercaptoethanol) and very high salt buffer (50 mM HEPES, pH = 7.5, 500 mM NaCl, 40 mM Imidazole, 5 mM β-mercaptoethanol).

Article Title: HIV-1 remodels the nuclear pore complex
Article Snippet: In brief, equal numbers of mock- and peak-infected T cells were collected, washed with PBS, incubated in hypotonic lysis buffer (10 mM Hepes, pH 7.4, 1.5 mM MgCl2 , and 10 mM KCl) for 10 min, Dounce homogenized (40-ml capacity Wheaton type; VWR), and resuspended in 2.2 M SHKM (2.2 M sucrose, 50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ). .. Pellets were resuspended in 0.25 M SHKM (50 mM Hepes, pH 7.4, 25 mM KCl, and 5 mM MgCl2 ), treated with RNase A and DNase I (Invitrogen) for 15 min, and resuspended in 10% SHM (0.3 M sucrose, 10 mM Hepes, pH 7.4, and 2 mM MgCl2 ) for centrifugation at 5,000 rpm for 10 min at 4°C.

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  • 77
    Thermo Fisher mouse anti human rnase l
    Activation of <t>RNase</t> L during WNV infection of HT1080 cells requires OAS3 expression. ( A ) OAS1-, OAS2-, OAS3-, and RNase L-KO HT1080 cells were mock-treated or were treated with IFN-α (2,000 U/mL) overnight. Proteins were analyzed by immunoblotting
    Mouse Anti Human Rnase L, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human rnase l/product/Thermo Fisher
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti human rnase l - by Bioz Stars, 2019-10
    77/100 stars
      Buy from Supplier

    81
    Thermo Fisher rnase a
    PO41 repeat is transcribed in chicken skeletal muscle cells.  FISH with PO41pos ( green , upper row) and PO41neg ( red , bottom row) probes on skeletal muscle cryosections.  (a, a’)  DNA/RNA hybridization revealed transcripts from both strands of PO41 repeat in cell nuclei.  (b, b’)  DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in all cell nuclei.  (c, c’)  RNase A treatment before DNA/RNA hybridization (negative control) removed all hybridization signals. Nuclei were counterstained with DAPI. Scale bars: 10 μm  (a) ; 20 μm  (b, c, a’) ; 30 μm  (b’, c’) .
    Rnase A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase a/product/Thermo Fisher
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    rnase a - by Bioz Stars, 2019-10
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    82
    Thermo Fisher gene exp rnase2 hs00795553 s1
    Expression of <t>EDN</t> and GATA-1 in response to overexpression of GATA-2. Relative expression of GATA-2 in uninduced HL-60 clone 15 cells 2 days after transduction with vector only ( pctrl ) or with the GATA-2 expression vector ( pGATA-2 ) was evaluated by
    Gene Exp Rnase2 Hs00795553 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp rnase2 hs00795553 s1/product/Thermo Fisher
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    gene exp rnase2 hs00795553 s1 - by Bioz Stars, 2019-10
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    Image Search Results


    Activation of RNase L during WNV infection of HT1080 cells requires OAS3 expression. ( A ) OAS1-, OAS2-, OAS3-, and RNase L-KO HT1080 cells were mock-treated or were treated with IFN-α (2,000 U/mL) overnight. Proteins were analyzed by immunoblotting

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Activation of RNase L during WNV infection of HT1080 cells requires OAS3 expression. ( A ) OAS1-, OAS2-, OAS3-, and RNase L-KO HT1080 cells were mock-treated or were treated with IFN-α (2,000 U/mL) overnight. Proteins were analyzed by immunoblotting

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Infection, Expressing, Gene Knockout

    Expression of OAS3 protein in OAS3-KO cells restores 2-5A production and RNase L activation. ( A ) OAS3-KO cells were transfected with p3XFlag-tagged OAS3 cDNA, and a cell line was generated by neomycin selection. Cell lysates were analyzed by electrophoresis

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Expression of OAS3 protein in OAS3-KO cells restores 2-5A production and RNase L activation. ( A ) OAS3-KO cells were transfected with p3XFlag-tagged OAS3 cDNA, and a cell line was generated by neomycin selection. Cell lysates were analyzed by electrophoresis

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Expressing, Gene Knockout, Activation Assay, Transfection, Generated, Selection, Electrophoresis

    Viruses that cause minimal or no activation of RNase L as determined by monitoring rRNA integrity in A549 cells. Parental and RNase L-KO cells were infected at MOI = 20. Cells were lysed at 12 (LACV, VSV, SeV, EMCV), 21 (TMEV), or 60 (LCMV) hpi, and RNA

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Viruses that cause minimal or no activation of RNase L as determined by monitoring rRNA integrity in A549 cells. Parental and RNase L-KO cells were infected at MOI = 20. Cells were lysed at 12 (LACV, VSV, SeV, EMCV), 21 (TMEV), or 60 (LCMV) hpi, and RNA

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Gene Knockout, Infection

    Activation of RNase L during SINV infection of A549 cells requires OAS3 expression. ( A ) WT A549 and KO cells were infected with SINV (MOI = 1 pfu per cell), and at 24 hpi cells were lysed and RNA integrity was assessed. The positions of 18S and 28S rRNA

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Activation of RNase L during SINV infection of A549 cells requires OAS3 expression. ( A ) WT A549 and KO cells were infected with SINV (MOI = 1 pfu per cell), and at 24 hpi cells were lysed and RNA integrity was assessed. The positions of 18S and 28S rRNA

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Infection, Expressing, Gene Knockout

    Activation of RNase L by pIC or VACVΔE3L in HT10810 or HME cells requires OAS3 expression. ( A and B ) HT1080 WT or KO cells were transfected with 500 ng/mL ( A ) or HME cells were transfected with1 μg/mL ( B ) of pIC, and at 4 h (HT1080 cells)

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Activation of RNase L by pIC or VACVΔE3L in HT10810 or HME cells requires OAS3 expression. ( A and B ) HT1080 WT or KO cells were transfected with 500 ng/mL ( A ) or HME cells were transfected with1 μg/mL ( B ) of pIC, and at 4 h (HT1080 cells)

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Expressing, Gene Knockout, Transfection

    IFN pretreatment reduces WNV titer in WT and OAS3-KO cells to the same level, independent of RNase L activation. ( A ) Cells were mock-treated or treated with 100 U of IFN-α overnight. Cells were transfected with 500 ng/mL of pIC or were infected

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: IFN pretreatment reduces WNV titer in WT and OAS3-KO cells to the same level, independent of RNase L activation. ( A ) Cells were mock-treated or treated with 100 U of IFN-α overnight. Cells were transfected with 500 ng/mL of pIC or were infected

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Gene Knockout, Activation Assay, Transfection, Infection

    Activation of RNase L by pIC in A549 cells requires OAS3 expression. ( A ) OAS1-, OAS2-, OAS3-, and RNase L-KO A549 cells were mock-treated or treated with IFN-β (1,000 U/mL) overnight. Cells were lysed, and proteins were analyzed by immunoblotting

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Activation of RNase L by pIC in A549 cells requires OAS3 expression. ( A ) OAS1-, OAS2-, OAS3-, and RNase L-KO A549 cells were mock-treated or treated with IFN-β (1,000 U/mL) overnight. Cells were lysed, and proteins were analyzed by immunoblotting

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Expressing, Gene Knockout

    Activation of RNase L during IAVΔNS1 infection of A549 cells requires OAS3 expression. ( A ) WT A549 and KO cells were infected with IAVΔNS1 (MOI = 10), and at 24 hpi RNA integrity was assessed. ( B ) Cells were infected with IAVΔNS1

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Activation of RNase L during IAVΔNS1 infection of A549 cells requires OAS3 expression. ( A ) WT A549 and KO cells were infected with IAVΔNS1 (MOI = 10), and at 24 hpi RNA integrity was assessed. ( B ) Cells were infected with IAVΔNS1

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Infection, Expressing, Gene Knockout

    Activation of RNase L during VACVΔE3L infection of A549 cells requires OAS3 expression. ( A ) WT A549 cells and KO cells were infected with VACVΔE3L (MOI = 10 pfu per cell), and at 24 hpi RNA integrity was assessed. ( B ) Cells were infected

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Activation of RNase L during VACVΔE3L infection of A549 cells requires OAS3 expression. ( A ) WT A549 cells and KO cells were infected with VACVΔE3L (MOI = 10 pfu per cell), and at 24 hpi RNA integrity was assessed. ( B ) Cells were infected

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Infection, Expressing, Gene Knockout

    Activation of RNase L during WNV infection of A549 cells requires OAS3 expression. ( A ) WT A549 and KO cells were infected with WNV (MOI = 5 pfu per cell), and at 24 hpi RNA integrity was assessed. ( B ) WT A549 and KO cells were infected with WNV (MOI =

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: Activation of RNase L during WNV infection of A549 cells requires OAS3 expression. ( A ) WT A549 and KO cells were infected with WNV (MOI = 5 pfu per cell), and at 24 hpi RNA integrity was assessed. ( B ) WT A549 and KO cells were infected with WNV (MOI =

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Activation Assay, Infection, Expressing, Gene Knockout

    The OAS–RNase L pathway. Upon detection of viral dsRNA during infection with diverse human viruses (WNV, SINV, IAVΔNS1, VACVΔE3L), OAS3 produces 2-5A, which activates RNase L. RNase L degrades cellular and viral RNA, leading to

    Journal:

    Article Title: Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses

    doi: 10.1073/pnas.1519657113

    Figure Lengend Snippet: The OAS–RNase L pathway. Upon detection of viral dsRNA during infection with diverse human viruses (WNV, SINV, IAVΔNS1, VACVΔE3L), OAS3 produces 2-5A, which activates RNase L. RNase L degrades cellular and viral RNA, leading to

    Article Snippet: OAS p42 is also referred to as “p41.” Mouse anti-OAS1 p41 F3 antibody (1:1,000; Santa Cruz), rabbit anti-OAS1 p42/46 D1W3A monoclonal antibody (1:200; Cell Signaling), rabbit anti-OAS2 H-180 polyclonal antibody (1:500; Santa Cruz), goat anti-OAS3 N-18 (1:250; Santa Cruz), mouse anti-human RNase L (1:1,000) , mouse anti-GAPDH GA1R (1:1,000; Thermo Fisher), and mouse anti-Flag M2 antibody (Sigma-Aldrich) were used to detect OAS1 p41, OAS1 p42/p46, OAS2 p69, OAS3 p100, RNase L, and GAPDH, respectively.

    Techniques: Infection

    PO41 repeat is transcribed in chicken skeletal muscle cells.  FISH with PO41pos ( green , upper row) and PO41neg ( red , bottom row) probes on skeletal muscle cryosections.  (a, a’)  DNA/RNA hybridization revealed transcripts from both strands of PO41 repeat in cell nuclei.  (b, b’)  DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in all cell nuclei.  (c, c’)  RNase A treatment before DNA/RNA hybridization (negative control) removed all hybridization signals. Nuclei were counterstained with DAPI. Scale bars: 10 μm  (a) ; 20 μm  (b, c, a’) ; 30 μm  (b’, c’) .

    Journal: Molecular Cytogenetics

    Article Title: Non-coding RNA derived from a conservative subtelomeric tandem repeat in chicken and Japanese quail somatic cells

    doi: 10.1186/s13039-014-0102-7

    Figure Lengend Snippet: PO41 repeat is transcribed in chicken skeletal muscle cells. FISH with PO41pos ( green , upper row) and PO41neg ( red , bottom row) probes on skeletal muscle cryosections. (a, a’) DNA/RNA hybridization revealed transcripts from both strands of PO41 repeat in cell nuclei. (b, b’) DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in all cell nuclei. (c, c’) RNase A treatment before DNA/RNA hybridization (negative control) removed all hybridization signals. Nuclei were counterstained with DAPI. Scale bars: 10 μm (a) ; 20 μm (b, c, a’) ; 30 μm (b’, c’) .

    Article Snippet: Samples for negative control were treated with RNase A (100 μg/ml, ThermoScientific).

    Techniques: Fluorescence In Situ Hybridization, Hybridization, DNA-DNA Hybridization, Positive Control, Negative Control

    PO41 repeat is transcribed in Japanese quail somatic cells.  FISH with PO41neg ( red ) probe on Japanese quail oviduct cryosections (bottom row).  (a)  DNA/RNA hybridization revealed transcripts of PO41 repeat in all cell layers.  (b)  RNase A treatment before DNA/RNA hybridization (negative control) removed nuclear hybridization signals. Nuclei were counterstained with DAPI. Scale bars: 40 μm.

    Journal: Molecular Cytogenetics

    Article Title: Non-coding RNA derived from a conservative subtelomeric tandem repeat in chicken and Japanese quail somatic cells

    doi: 10.1186/s13039-014-0102-7

    Figure Lengend Snippet: PO41 repeat is transcribed in Japanese quail somatic cells. FISH with PO41neg ( red ) probe on Japanese quail oviduct cryosections (bottom row). (a) DNA/RNA hybridization revealed transcripts of PO41 repeat in all cell layers. (b) RNase A treatment before DNA/RNA hybridization (negative control) removed nuclear hybridization signals. Nuclei were counterstained with DAPI. Scale bars: 40 μm.

    Article Snippet: Samples for negative control were treated with RNase A (100 μg/ml, ThermoScientific).

    Techniques: Fluorescence In Situ Hybridization, Hybridization, Negative Control

    Sensitivity of G-rich PO41 transcripts during the cell cycle to RNases with different substrate specificity.  Treatment of MDCC-MSB1 cells with different RNases before or after (RNase H*) DNA/RNA FISH with PO41neg ( red ) probes. MDCC-MSB1 cells are shown at interphase, prophase, metaphase, anaphase and telophase stages. RNases used for treatments are indicated on the left. PO41 RNA was degraded by RiboShredder RNases cocktail, RNase A treatments and RNase H treatment performed after FISH. RNase H and RNase III treatments completely removed only dispersed G-rich transcripts at interphase and decreased the intensity of RNA FISH signals at other stages of cell cycle. DNA was counterstained with DAPI. Scale bar: 5 μm.

    Journal: Molecular Cytogenetics

    Article Title: Non-coding RNA derived from a conservative subtelomeric tandem repeat in chicken and Japanese quail somatic cells

    doi: 10.1186/s13039-014-0102-7

    Figure Lengend Snippet: Sensitivity of G-rich PO41 transcripts during the cell cycle to RNases with different substrate specificity. Treatment of MDCC-MSB1 cells with different RNases before or after (RNase H*) DNA/RNA FISH with PO41neg ( red ) probes. MDCC-MSB1 cells are shown at interphase, prophase, metaphase, anaphase and telophase stages. RNases used for treatments are indicated on the left. PO41 RNA was degraded by RiboShredder RNases cocktail, RNase A treatments and RNase H treatment performed after FISH. RNase H and RNase III treatments completely removed only dispersed G-rich transcripts at interphase and decreased the intensity of RNA FISH signals at other stages of cell cycle. DNA was counterstained with DAPI. Scale bar: 5 μm.

    Article Snippet: Samples for negative control were treated with RNase A (100 μg/ml, ThermoScientific).

    Techniques: Fluorescence In Situ Hybridization

    PO41 repeat is transcribed in chicken somatic tissues. (a-d)  3D DNA/RNA FISH with PO41neg probe ( red ) on whole mount fragments of chicken somatic tissues revealed PO41 repeat transcripts in cell nuclei of muscles  (a) , brain  (b, d)  and oviduct  (c) .  (e)  3D DNA/DNA FISH (positive control) and  (f)  3D DNA/RNA FISH after RNase A treatment (negative control) are shown for fragments of cerebellum. Nuclei were counterstained with DAPI. Scale bars: 10 μm  (a, b, d, f) , 20 μm  (e, c) .

    Journal: Molecular Cytogenetics

    Article Title: Non-coding RNA derived from a conservative subtelomeric tandem repeat in chicken and Japanese quail somatic cells

    doi: 10.1186/s13039-014-0102-7

    Figure Lengend Snippet: PO41 repeat is transcribed in chicken somatic tissues. (a-d) 3D DNA/RNA FISH with PO41neg probe ( red ) on whole mount fragments of chicken somatic tissues revealed PO41 repeat transcripts in cell nuclei of muscles (a) , brain (b, d) and oviduct (c) . (e) 3D DNA/DNA FISH (positive control) and (f) 3D DNA/RNA FISH after RNase A treatment (negative control) are shown for fragments of cerebellum. Nuclei were counterstained with DAPI. Scale bars: 10 μm (a, b, d, f) , 20 μm (e, c) .

    Article Snippet: Samples for negative control were treated with RNase A (100 μg/ml, ThermoScientific).

    Techniques: Fluorescence In Situ Hybridization, Positive Control, Negative Control

    C-rich transcripts of PO41 repeat in chicken small intestine cells.  FISH with PO41pos ( green , upper row) and PO41neg ( red , bottom row) probes on small intestine cryosections.  (a, a’)  DNA/RNA hybridization revealed C-rich ( green ), but not G-rich ( red ) PO41 repeat transcripts in mucous membrane of chicken small intestine.  (b, b’)  DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in all cell nuclei.  (c, c’)  RNase A treatment before DNA/RNA hybridization (negative control) removed hybridization signals with PO41pos probe from the cell nuclei, but didn’t remove signals in cytoplasmic granules within the mucous membrane. Right-hand images demonstrate enlarged fragments of muscles layer (upper panels) and mucous membrane (bottom panels) of small intestine. Nuclei were counterstained with DAPI. Scale bar: 40 μm.

    Journal: Molecular Cytogenetics

    Article Title: Non-coding RNA derived from a conservative subtelomeric tandem repeat in chicken and Japanese quail somatic cells

    doi: 10.1186/s13039-014-0102-7

    Figure Lengend Snippet: C-rich transcripts of PO41 repeat in chicken small intestine cells. FISH with PO41pos ( green , upper row) and PO41neg ( red , bottom row) probes on small intestine cryosections. (a, a’) DNA/RNA hybridization revealed C-rich ( green ), but not G-rich ( red ) PO41 repeat transcripts in mucous membrane of chicken small intestine. (b, b’) DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in all cell nuclei. (c, c’) RNase A treatment before DNA/RNA hybridization (negative control) removed hybridization signals with PO41pos probe from the cell nuclei, but didn’t remove signals in cytoplasmic granules within the mucous membrane. Right-hand images demonstrate enlarged fragments of muscles layer (upper panels) and mucous membrane (bottom panels) of small intestine. Nuclei were counterstained with DAPI. Scale bar: 40 μm.

    Article Snippet: Samples for negative control were treated with RNase A (100 μg/ml, ThermoScientific).

    Techniques: Fluorescence In Situ Hybridization, Hybridization, DNA-DNA Hybridization, Positive Control, Negative Control

    Expression of EDN and GATA-1 in response to overexpression of GATA-2. Relative expression of GATA-2 in uninduced HL-60 clone 15 cells 2 days after transduction with vector only ( pctrl ) or with the GATA-2 expression vector ( pGATA-2 ) was evaluated by

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Expression of EDN and GATA-1 in response to overexpression of GATA-2. Relative expression of GATA-2 in uninduced HL-60 clone 15 cells 2 days after transduction with vector only ( pctrl ) or with the GATA-2 expression vector ( pGATA-2 ) was evaluated by

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Expressing, Over Expression, Transduction, Plasmid Preparation

    Expression of EDN and GATA-1 in response to lentivirus-mediated suppression of GATA-2 in differentiating eosinophils from CD34 + hematopoietic progenitors. A, modified Giemsa-stained cytospin preparations of cells differentiating in eosinophilopoietic

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Expression of EDN and GATA-1 in response to lentivirus-mediated suppression of GATA-2 in differentiating eosinophils from CD34 + hematopoietic progenitors. A, modified Giemsa-stained cytospin preparations of cells differentiating in eosinophilopoietic

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Expressing, Modification, Staining

    Contribution of GATA consensus binding sites to the transcription of the EDN gene. A, schematic of the gene encoding eosinophil-derived neurotoxin/ RNase 2 indicating relative positions of consensus binding sites for GATA family transcription factors.

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Contribution of GATA consensus binding sites to the transcription of the EDN gene. A, schematic of the gene encoding eosinophil-derived neurotoxin/ RNase 2 indicating relative positions of consensus binding sites for GATA family transcription factors.

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Binding Assay, Derivative Assay

    Electrophoretic mobility shift/supershift assays for evaluating interactions of consensus GATA sequences in the 5 ′ promoter of EDN with BA-treated HL-60 clone 15 nuclear extracts and anti-GATA-1 and anti-GATA-2 antibodies. A, lane 1 , biotin-labeled

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Electrophoretic mobility shift/supershift assays for evaluating interactions of consensus GATA sequences in the 5 ′ promoter of EDN with BA-treated HL-60 clone 15 nuclear extracts and anti-GATA-1 and anti-GATA-2 antibodies. A, lane 1 , biotin-labeled

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Electrophoretic Mobility Shift Assay, Labeling

    Silencing of GATA-1 and its impact on EDN expression. A, transcription of GATA-1 in BA-differentiated HL-60 clone 15 cells transfected with irrelevant control sequence or a GATA-1-directed oligonucleotide was evaluated by quantitative RT-PCR. B, transcription

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Silencing of GATA-1 and its impact on EDN expression. A, transcription of GATA-1 in BA-differentiated HL-60 clone 15 cells transfected with irrelevant control sequence or a GATA-1-directed oligonucleotide was evaluated by quantitative RT-PCR. B, transcription

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Expressing, Transfection, Sequencing, Quantitative RT-PCR

    Chromatin immunoprecipitation assay for evaluating interactions of consensus GATA sequences in the 5 ′ promoter of EDN . A, PCR amplification of the EDN GATA -1114 sequence from the following: lane 1 , unmanipulated genomic DNA; lane 2 , cross-linked

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Chromatin immunoprecipitation assay for evaluating interactions of consensus GATA sequences in the 5 ′ promoter of EDN . A, PCR amplification of the EDN GATA -1114 sequence from the following: lane 1 , unmanipulated genomic DNA; lane 2 , cross-linked

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing

    Expression of transcripts encoding EDN ( A ), GATA-1 ( B ), and GATA-2 ( C ) in the HL-60 clone 15 eosinophil promyelocyte cell line in response to BA-induced differentiation. Cells were induced to differentiate with 0.5 m m BA under alkaline conditions,

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Expression of transcripts encoding EDN ( A ), GATA-1 ( B ), and GATA-2 ( C ) in the HL-60 clone 15 eosinophil promyelocyte cell line in response to BA-induced differentiation. Cells were induced to differentiate with 0.5 m m BA under alkaline conditions,

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Expressing

    Silencing of GATA-2 and its impact on EDN expression. A, transcription of GATA-2 in BA-differentiated HL-60 clone 15 cells transfected with control (irrelevant sequence) or one of two independent GATA-2-directed oligonucleotides was evaluated by quantitative

    Journal:

    Article Title: GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

    doi: 10.1074/jbc.M807307200

    Figure Lengend Snippet: Silencing of GATA-2 and its impact on EDN expression. A, transcription of GATA-2 in BA-differentiated HL-60 clone 15 cells transfected with control (irrelevant sequence) or one of two independent GATA-2-directed oligonucleotides was evaluated by quantitative

    Article Snippet: GATA-2 assay identification was Hs00927739_m1, and probe was 5′-CCC ACG GCT GCG TGT GGC CGT TGC C-3′; EDN assay identification was Hs00795553_s1, and probe was 5′-TCA CCA CAG TGG AAG CCA GGT GCC T-3′; GATA-1 assay identification was Hs00231112_m1, and probe was 5′-CAC CAG CCC AGG TTA ATC CCC AGA G-3′; the probes were labeled with FAM and NFQ.

    Techniques: Expressing, Transfection, Sequencing