dnase i Thermo Fisher Search Results


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  • 99
    Thermo Fisher dnase i enzyme
    <t>DNase</t> I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Dnase I Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnase i
    <t>DNase</t> I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dnase i
    Effects of noncomplementary dNTPs on <t>DNase</t> I footprints and stable complex formation by HIV-1 RT
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 60695 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase free dnase i
    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from <t>RNase</t> A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or <t>DNase</t> I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p
    Rnase Free Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher a594 dnaase i
    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from <t>RNase</t> A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or <t>DNase</t> I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p
    A594 Dnaase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher turbo dnase i
    α-subunits of RNAP bind to A-boxes upstream of the T-tract. A) DNA sequence of the P sabA upstream region showing the predicted UP-like elements and multiple A-boxes (red boxes). Red, blue and green lines mark the interaction sites of σ 70 -RNAP found by Footprint analysis, correspondingly, see Fig. 5B–C . B–C) Mapping of the binding site for σ 70 -RNAP to P sabA DNA using <t>DNase</t> I footprint assay. 10 nM of [γ 32 P]ATP-labeled P sabA DNA (−166 to +74) were mixed with increasing concentrations of σ 70 -RNAP (0, 6.25, 12.5, 25, or 50 nM). The regions protected from DNase I cleavage are marked by red (core promoter), blue (proximal UP-like element) and green (distal UP-like element) lines. The positions of the T-tract, predicted −35 and −10, and +1 transcriptional start site, are indicated to the left. The stars mark the region of the promoter that was deleted in Δ 46 variants (−97 to −49, see also Fig. S2 and S6A ). Nucleotide positions, relative to the transcriptional start site, are shown to the right. D) Binding of σ 70 -RNAP (55 nM) to P sabA DNA (−166 to +74), with different repeat tract compositions and promoter mutant variants, analyzed by SPR. The sensorgrams show values normalized to that of the full-length T 13 -variant. Binding to a sabA CDS-fragment, also used in Fig. 4 , is shown as a background curve in the top diagram. The bottom diagram is an enlargement of the dotted-lined square in the top diagram. E) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing P sabA with proximal UP-like element deleted. The constructs contain different tract lengths and compositions (see Fig. 5B–C and S6A ). Black bars represent wt promoters and white bars Δ 46 variants, respectively. β-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods . Data is presented as relative values with activity of P sabA T 13 wt set to 1. F) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing sabA promoter with scrambled UP-like elements. β-galactosidase assays were performed as described in Fig. 5E and data is presented as relative values with activity of P sabA wt set to 1.
    Turbo Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher ambion dnase i
    α-subunits of RNAP bind to A-boxes upstream of the T-tract. A) DNA sequence of the P sabA upstream region showing the predicted UP-like elements and multiple A-boxes (red boxes). Red, blue and green lines mark the interaction sites of σ 70 -RNAP found by Footprint analysis, correspondingly, see Fig. 5B–C . B–C) Mapping of the binding site for σ 70 -RNAP to P sabA DNA using <t>DNase</t> I footprint assay. 10 nM of [γ 32 P]ATP-labeled P sabA DNA (−166 to +74) were mixed with increasing concentrations of σ 70 -RNAP (0, 6.25, 12.5, 25, or 50 nM). The regions protected from DNase I cleavage are marked by red (core promoter), blue (proximal UP-like element) and green (distal UP-like element) lines. The positions of the T-tract, predicted −35 and −10, and +1 transcriptional start site, are indicated to the left. The stars mark the region of the promoter that was deleted in Δ 46 variants (−97 to −49, see also Fig. S2 and S6A ). Nucleotide positions, relative to the transcriptional start site, are shown to the right. D) Binding of σ 70 -RNAP (55 nM) to P sabA DNA (−166 to +74), with different repeat tract compositions and promoter mutant variants, analyzed by SPR. The sensorgrams show values normalized to that of the full-length T 13 -variant. Binding to a sabA CDS-fragment, also used in Fig. 4 , is shown as a background curve in the top diagram. The bottom diagram is an enlargement of the dotted-lined square in the top diagram. E) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing P sabA with proximal UP-like element deleted. The constructs contain different tract lengths and compositions (see Fig. 5B–C and S6A ). Black bars represent wt promoters and white bars Δ 46 variants, respectively. β-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods . Data is presented as relative values with activity of P sabA T 13 wt set to 1. F) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing sabA promoter with scrambled UP-like elements. β-galactosidase assays were performed as described in Fig. 5E and data is presented as relative values with activity of P sabA wt set to 1.
    Ambion Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dnase i buffer
    Analysis of the Mur34 binding site by <t>DNase</t> I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    Dnase I Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher m v dnase i
    Analysis of the Mur34 binding site by <t>DNase</t> I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    M V Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher deoxyribonuclease
    Detection of MDV transcripts in BMMs and BMDCs infected with MDV in vitro . BMMs and BMDCs were infected in vitro with EGFP-expressing MDV. After 3 days, EGFP-positive cells were sorted and RT-PCR was carried out for the detection of (a) immediate early ICP4 (200 bp), (b) early pp38 (198 bp), (c) late gB (193 bp) and (d) MDV-specific l -Meq (200 bp) transcripts. L, ladder; +, positive control MDV-infected CEFs; −, negative control, nuclease-free H 2 O; M, infected BMMs (cDNA); MN, infected BMMs no-RT control <t>(DNase-treated</t> <t>RNA);</t> D, infected BMDCs (cDNA); DN, infected BMDCs no-RT control (DNase-treated RNA).
    Deoxyribonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gt dnase i
    Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with <t>DNase</t> I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.
    Gt Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i rnasefree
    Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with <t>DNase</t> I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.
    Dnase I Rnasefree, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Staining, Standard Deviation, MANN-WHITNEY

    DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Incubation, Staining, Concentration Assay, Standard Deviation, MANN-WHITNEY

    Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Inhibition, Incubation, Standard Deviation, MANN-WHITNEY

    Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Incubation, Standard Deviation, MANN-WHITNEY

    Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    DNase I protection on P/Ts terminated with dT analogues

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: DNase I protection on P/Ts terminated with dT analogues

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

    Journal:

    Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

    doi: 10.1016/j.jmb.2007.03.006

    Figure Lengend Snippet: Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

    Article Snippet: After incubation on ice for 5 min, 0.03 U DNase I (USB corp.), in RB buffer, was added and the samples were incubated at room temperature for 3 min.

    Techniques:

    DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay, Functional Assay

    The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    Analysis of RNA isolated from purified virions and dense bodies (DBs). Virions and DBs were purified from RNase-ONE-treated virus stocks, dialysed and treated with micrococcal nuclease; RNA was extracted, and during the RNA extraction the column was treated with DNase-I. (a) RNA concentrations of virions and DBs. Presence of (b) viral IE RNA transcripts and (c) lncRNA2.7 transcripts determined by TaqMan PCR assays. Values are mean± sem of three experiments performed in triplicate. RT, reverse transcriptase.

    Journal: The Journal of General Virology

    Article Title: Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

    doi: 10.1099/jgv.0.000736

    Figure Lengend Snippet: Analysis of RNA isolated from purified virions and dense bodies (DBs). Virions and DBs were purified from RNase-ONE-treated virus stocks, dialysed and treated with micrococcal nuclease; RNA was extracted, and during the RNA extraction the column was treated with DNase-I. (a) RNA concentrations of virions and DBs. Presence of (b) viral IE RNA transcripts and (c) lncRNA2.7 transcripts determined by TaqMan PCR assays. Values are mean± sem of three experiments performed in triplicate. RT, reverse transcriptase.

    Article Snippet: RNA was isolated with the miRNeasy mini kit (Qiagen), including the DNase-I treatment or isolated with Trizol-LS reagent (Life Technologies).

    Techniques: Isolation, Purification, RNA Extraction, Polymerase Chain Reaction

    Procedure to study protein-nucleic acid interactions. The oligonucleotide is designed to contain a 5'-biotin tag, a 3'-fluorescent tag and a UV sensitive group in the middle. (A) Crosslink initiated by exposing to UV (305+16 nm). (B) After three hours of UV activation, denatured samples were subjected to SDS-PAGE analysis, where crosslinked species were confirmed by fluorescent imaging and all bands were visualized by Commassie staining technique. (C) Interesting spots were picked for protease enzymatic in-gel digestion to yield peptide mixtures. (D) Crosslinked peptides were extracted by magnetic streptavidin beads and uncrosslinked peptides were washed away. (E) The crosslinked peptides were subjected to DNase I degradation to minimize the attaching oligonucleotide moieties. (F) Crosslinked peptides with the remaining nucleic acid attached were extracted by reverse phase ZipTip C18 cartridge and analyzed by Q-tof ESI. (G) Raw data were collected and processed by Protein-Lynx to generate a PKL file, which was used as input to CLPM to identify matches with theoretical peptides.

    Journal: BMC Bioinformatics

    Article Title: CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

    doi: 10.1186/1471-2105-6-S2-S9

    Figure Lengend Snippet: Procedure to study protein-nucleic acid interactions. The oligonucleotide is designed to contain a 5'-biotin tag, a 3'-fluorescent tag and a UV sensitive group in the middle. (A) Crosslink initiated by exposing to UV (305+16 nm). (B) After three hours of UV activation, denatured samples were subjected to SDS-PAGE analysis, where crosslinked species were confirmed by fluorescent imaging and all bands were visualized by Commassie staining technique. (C) Interesting spots were picked for protease enzymatic in-gel digestion to yield peptide mixtures. (D) Crosslinked peptides were extracted by magnetic streptavidin beads and uncrosslinked peptides were washed away. (E) The crosslinked peptides were subjected to DNase I degradation to minimize the attaching oligonucleotide moieties. (F) Crosslinked peptides with the remaining nucleic acid attached were extracted by reverse phase ZipTip C18 cartridge and analyzed by Q-tof ESI. (G) Raw data were collected and processed by Protein-Lynx to generate a PKL file, which was used as input to CLPM to identify matches with theoretical peptides.

    Article Snippet: • DNase I (Ambion).

    Techniques: Activation Assay, SDS Page, Imaging, Staining

    Possible ion structures for the fragmentation of tryptic peptide crosslinked to the dinucleotide (dGdU) after DNase I digestion as proposed by Golden et al [31] . The blue line on the right of each diagram represents the peptide moiety in the heteroconjugate.

    Journal: BMC Bioinformatics

    Article Title: CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

    doi: 10.1186/1471-2105-6-S2-S9

    Figure Lengend Snippet: Possible ion structures for the fragmentation of tryptic peptide crosslinked to the dinucleotide (dGdU) after DNase I digestion as proposed by Golden et al [31] . The blue line on the right of each diagram represents the peptide moiety in the heteroconjugate.

    Article Snippet: • DNase I (Ambion).

    Techniques:

    DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Journal: The Journal of Pathology: Clinical Research

    Article Title: Lupus nephritis: low urinary DNase I levels reflect loss of renal DNase I and may be utilized as a biomarker of disease progression

    doi: 10.1002/cjp2.99

    Figure Lengend Snippet: DNase I expression and activity. (A) The protein expression pattern of DNase I in kidney sections from (NZBxNZW)F1 mice as revealed by IHC, IF, IEM, and confocal microscopy (DNase I in red and Trap1 in green as a cytoplasmic marker). (B) DNase I endonuclease activity as revealed by zymography on samples from whole kidney lysates of representative Group 1, 2, and 3 (NZBxNZW)F1 mice, as well as a control (R, recombinant DNase I) and a serum (S) sample. (C) Urinary DNase I endonuclease activity in different groups of (NZBxNZW)F1 mice as revealed by zymography.

    Article Snippet: In addition, a radial diffusion assay was used to measure DNase I activity in 1% agarose gel with 30 µg/ml heat‐denatured salmon sperm DNA (Invitrogen) in the DNase I reaction buffer.

    Techniques: Expressing, Activity Assay, Mouse Assay, Immunohistochemistry, Confocal Microscopy, Marker, Zymography, Recombinant

    Kinetics of loop DNA digestion with DNase I in nucleoids from P0, P7, P80 and P540 rat neurons. Nucleoids were treated with DNase I at 0.92 U/ml. Each time-point value corresponds to the average of independent experiments using separate animals as the source of nucleoids (P0 n = 4; P7 n = 5; P80 n = 5; P540 n = 4). The data sets for P7 and P540 were taken from our previous work [21] . The topological zones relative to the NM for the corresponding kinetics were established considering the local slopes between pairs of time points ( Table 3 ) and the corresponding S.D.

    Journal: PLoS ONE

    Article Title: Continued Stabilization of the Nuclear Higher-Order Structure of Post-Mitotic Neurons In Vivo

    doi: 10.1371/journal.pone.0021360

    Figure Lengend Snippet: Kinetics of loop DNA digestion with DNase I in nucleoids from P0, P7, P80 and P540 rat neurons. Nucleoids were treated with DNase I at 0.92 U/ml. Each time-point value corresponds to the average of independent experiments using separate animals as the source of nucleoids (P0 n = 4; P7 n = 5; P80 n = 5; P540 n = 4). The data sets for P7 and P540 were taken from our previous work [21] . The topological zones relative to the NM for the corresponding kinetics were established considering the local slopes between pairs of time points ( Table 3 ) and the corresponding S.D.

    Article Snippet: Standard PCR was carried out using 1.25 U GoTaq Flexi DNA polymerase (Promega) and 60 ng of nuclear matrix-bound DNA obtained from each DNase I digestion point, using an Applied Biosystems 2720 thermal cycler, and the same amplification program was used for all pairs of primers ( ): initial denaturising step at 94°C for 5 min, denaturising step at 94°C for 45 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min for 35 cycles, with a final extension at 72°C for 10 min.

    Techniques:

    Assessment by RT-PCR of puro r and hTERT transgene expression following transduction of Schistosoma japonicum schistosomules by pBABE-puro-hTERT retrovirus Total RNA samples were extracted from schistosomules and treated with DNase I. End point PCR was performed using reverse transcribed RNA/cDNA. The α-tubulin gene, amplified as an internal control to verify the integrity of the schistosome RNA and equivalent loadings of cDNAs, produced a 399 bp amplicon of the expected size. (A) Amplification using primers specific for the puro r gene; (B) Amplification using primers specific for the hTERT gene. Lanes: M, molecular size markers; 1, water, negative control; 2, RNA not reverse transcribed into cDNA from schistosomules transduced by virions; 3, cDNA from non-virus exposed schistosomules; 4, cDNA from schistosomules transduced by virions; P, positive control, pBABE- puro-hTERT plasmid.

    Journal: Molecular and biochemical parasitology

    Article Title: Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes

    doi: 10.1016/j.molbiopara.2010.07.007

    Figure Lengend Snippet: Assessment by RT-PCR of puro r and hTERT transgene expression following transduction of Schistosoma japonicum schistosomules by pBABE-puro-hTERT retrovirus Total RNA samples were extracted from schistosomules and treated with DNase I. End point PCR was performed using reverse transcribed RNA/cDNA. The α-tubulin gene, amplified as an internal control to verify the integrity of the schistosome RNA and equivalent loadings of cDNAs, produced a 399 bp amplicon of the expected size. (A) Amplification using primers specific for the puro r gene; (B) Amplification using primers specific for the hTERT gene. Lanes: M, molecular size markers; 1, water, negative control; 2, RNA not reverse transcribed into cDNA from schistosomules transduced by virions; 3, cDNA from non-virus exposed schistosomules; 4, cDNA from schistosomules transduced by virions; P, positive control, pBABE- puro-hTERT plasmid.

    Article Snippet: Total RNA was isolated from the worms using Trizol Reagent (Gibco-BRL) according to the manufacturer’s protocol, and the RNA incubated with RNase-free DNase I in the presence of Ribonuclease inhibitor (Fermentas, Burlington, Canada) to remove any residual, contaminating genomic DNA (gDNA). cDNA synthesis was accomplished using 5 μg DNase I-treated RNA, 1 μl random primer, 2 μl dNTPs, and 200 U M-MuLV Reverse Transcriptase using the First Strand cDNA Synthesis Kit (Fermentas), and the hTERT and puror genes amplified using the primers described above.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transduction, Polymerase Chain Reaction, Amplification, Produced, Negative Control, Positive Control, Plasmid Preparation

    RhA3A induces low frequencies of G-to-A mutations in SHIVΔ vif nascent reverse transcripts. TZM-Bl cells were infected with DNAse-I treated SHIVΔ vif or SHIV virions and DNA was extracted after 24 h. A 300-bp segment of nef was amplified,

    Journal: Virology

    Article Title: Differential virus restriction patterns of rhesus macaque and human APOBEC3A: implications for lentivirus evolution

    doi: 10.1016/j.virol.2011.07.017

    Figure Lengend Snippet: RhA3A induces low frequencies of G-to-A mutations in SHIVΔ vif nascent reverse transcripts. TZM-Bl cells were infected with DNAse-I treated SHIVΔ vif or SHIV virions and DNA was extracted after 24 h. A 300-bp segment of nef was amplified,

    Article Snippet: The resulting supernatant was DNase-I-treated (Fermentas) at 37°C for 30 min to minimize plasmid carry-over.

    Techniques: Infection, Amplification

    Luciferase gene expression and DNA accessibility as a function of r charge using pre-casted RNA gels. (A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the in vitro transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Luciferase gene expression and DNA accessibility as a function of r charge using pre-casted RNA gels. (A) G4 dendrimers and (B) CTAB. The synthesized amounts of RNA are displayed and samples were not pretreated with Dnase I. References are displayed in B where lane 1 shows the sample consisting only of DNA and without any compacting agent or transcriptional activity. Lane 2 shows the control sample containing DNA and the in vitro transcription mixture in the absence of compacting agents. Gels were post-stained using GelStar.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Luciferase, Expressing, Synthesized, Activity Assay, In Vitro, Staining

    Protection of DNA against Dnase I digestion by CTAB using a gel stained with EtBr. A gel electrophoresis gel where samples in lanes 1 and 6 contain linearized plasmid DNA only (control). The remaining lanes contain CTAB/DNA of the r charge values indicated. The samples loaded onto lanes 6–10 were incubated with Dnase I for 20 min following DNA condensation. At r charge ≤1.0, the DNA is completely degraded. At higher concentrations of CTAB, DNA degradation is inhibited.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Protection of DNA against Dnase I digestion by CTAB using a gel stained with EtBr. A gel electrophoresis gel where samples in lanes 1 and 6 contain linearized plasmid DNA only (control). The remaining lanes contain CTAB/DNA of the r charge values indicated. The samples loaded onto lanes 6–10 were incubated with Dnase I for 20 min following DNA condensation. At r charge ≤1.0, the DNA is completely degraded. At higher concentrations of CTAB, DNA degradation is inhibited.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Staining, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation

    Protection of DNA against Dnase I digestion using gels stained with GelStar. (A) G4/DNA complexes with r charge = 0.4 are used and the untreated complex is loaded on lane 1. The 2 nd lane displays the dissociated complex after treatment with 10 μg mL −1 heparin for 30 min. All other samples (lanes 3–7) are treated with 1 unit of Dnase I for the indicated time periods. To the samples in lanes 4–7, heparin was added after the Dnase I enzyme was heat inactivated. (B) Linearized plasmid DNA only is loaded onto lane 1 and the sample loaded onto lane 2 contains DNA, treated with Dnase I for 30 min. Samples loaded onto lanes 3–7 contain DNA and CTAB ( r charge = 7.5). Samples loaded onto lanes 4–7 are treated with Dnase I for the time periods indicated.

    Journal: PLoS ONE

    Article Title: DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation

    doi: 10.1371/journal.pone.0092692

    Figure Lengend Snippet: Protection of DNA against Dnase I digestion using gels stained with GelStar. (A) G4/DNA complexes with r charge = 0.4 are used and the untreated complex is loaded on lane 1. The 2 nd lane displays the dissociated complex after treatment with 10 μg mL −1 heparin for 30 min. All other samples (lanes 3–7) are treated with 1 unit of Dnase I for the indicated time periods. To the samples in lanes 4–7, heparin was added after the Dnase I enzyme was heat inactivated. (B) Linearized plasmid DNA only is loaded onto lane 1 and the sample loaded onto lane 2 contains DNA, treated with Dnase I for 30 min. Samples loaded onto lanes 3–7 contain DNA and CTAB ( r charge = 7.5). Samples loaded onto lanes 4–7 are treated with Dnase I for the time periods indicated.

    Article Snippet: Degradation of DNA Dnase I (Turbo Dnase I, Ambion) was used to elucidate how the degree of compaction affects the protection against enzymatic digestion of DNA.

    Techniques: Staining, Plasmid Preparation

    Developing surface-tethered nuclease sensor (SNS) for the in situ mapping of membrane-bound nuclease (MN) activity on the cell membrane. (A) SNS is a fluorophore (here Cy3) conjugated with a biotin and a quencher-labeled dsDNA. The fluorophore is freed from quenching when the dsDNA is degraded by MN, thus reporting the MN activity by fluorescence on site. (B) SNS-coated surface reported DNase I in solution at a series of concentrations. (C) The detection limit for soluble DNase I by SNS is calibrated to be 0.01 unit/ml (rounding 0.05/8.3 to 0.01). (D) An SNS-coated surface reported highly organized MN activity on the ventral membrane of adherent MDA-MB-231 cells. (E) The structure feature of the SNS pattern is finer than 1 μm.

    Journal: Journal of biophotonics

    Article Title: Optical sensor revealed abnormal nuclease spatial activity on cancer cell membrane

    doi: 10.1002/jbio.201800351

    Figure Lengend Snippet: Developing surface-tethered nuclease sensor (SNS) for the in situ mapping of membrane-bound nuclease (MN) activity on the cell membrane. (A) SNS is a fluorophore (here Cy3) conjugated with a biotin and a quencher-labeled dsDNA. The fluorophore is freed from quenching when the dsDNA is degraded by MN, thus reporting the MN activity by fluorescence on site. (B) SNS-coated surface reported DNase I in solution at a series of concentrations. (C) The detection limit for soluble DNase I by SNS is calibrated to be 0.01 unit/ml (rounding 0.05/8.3 to 0.01). (D) An SNS-coated surface reported highly organized MN activity on the ventral membrane of adherent MDA-MB-231 cells. (E) The structure feature of the SNS pattern is finer than 1 μm.

    Article Snippet: Soluble nuclease DNase I (89836, ThermoFisher Scientific) solutions at a series of concentrations were added to SNS coated petridishes.

    Techniques: In Situ, Activity Assay, Labeling, Fluorescence, Multiple Displacement Amplification

    Minor satellite RNA associate with CENP-A-containing chromatin. ( A ) RNA NChIP from nuclear extracts of asynchronously growing (AS) or nocodazole-arrested (NOC) MEL cells using antibodies against CENP-A, H3K9Me3 or H3Ac. Following DNaseI digestion, minor satellite RNA (minsat) and rDNA transcripts (rDNA) were analysed by RT-PCR, from the immunoprecipitated (IP) or supernatant fractions (SUP), in reactions containing (+) or not (−) RT. Control reactions in the absence of immunoprecipitating antibody (beads) were used as a negative control. ( B ) RNA pull-down experiments using in vitro transcribed minor satellite RNA (minsatF), followed by western blot (WB) analysis with the indicated antibodies. As a control, a similar experiment was performed with in vitro transcribed minor satellite RNA from the reverse strand (minsatR), therefore not complementary to the biotinylated DNA/LNA oligonucleotide used, or in the absence of RNA.

    Journal: Nucleic Acids Research

    Article Title: Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase

    doi: 10.1093/nar/gkp529

    Figure Lengend Snippet: Minor satellite RNA associate with CENP-A-containing chromatin. ( A ) RNA NChIP from nuclear extracts of asynchronously growing (AS) or nocodazole-arrested (NOC) MEL cells using antibodies against CENP-A, H3K9Me3 or H3Ac. Following DNaseI digestion, minor satellite RNA (minsat) and rDNA transcripts (rDNA) were analysed by RT-PCR, from the immunoprecipitated (IP) or supernatant fractions (SUP), in reactions containing (+) or not (−) RT. Control reactions in the absence of immunoprecipitating antibody (beads) were used as a negative control. ( B ) RNA pull-down experiments using in vitro transcribed minor satellite RNA (minsatF), followed by western blot (WB) analysis with the indicated antibodies. As a control, a similar experiment was performed with in vitro transcribed minor satellite RNA from the reverse strand (minsatR), therefore not complementary to the biotinylated DNA/LNA oligonucleotide used, or in the absence of RNA.

    Article Snippet: Genomic DNA was removed by digestion with 2 U of DNaseI (Ambion). cDNA were synthesized from an equivalent of 500 000 cells ( A) or 100 000 cells ( B and C), using random hexamers and Superscript II reverse transcriptase (Invitrogen), and amplified by PCR.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Negative Control, In Vitro, Western Blot

    The unmethylated human IGF2R intron 2 CpG island contains a DNase1 hypersensitive site. (A) Map of human IGF2R intron 2 (17.686 kb) showing the direction of IGF2R transcription. A map of the fragment used to generate the mouse transgenes is shown underneath.

    Journal: Genomics

    Article Title: Identification of the human homolog of the imprinted mouse Air non-coding RNA

    doi: 10.1016/j.ygeno.2008.08.004

    Figure Lengend Snippet: The unmethylated human IGF2R intron 2 CpG island contains a DNase1 hypersensitive site. (A) Map of human IGF2R intron 2 (17.686 kb) showing the direction of IGF2R transcription. A map of the fragment used to generate the mouse transgenes is shown underneath.

    Article Snippet: 20 μg of total DNase1 (DNA-freeTM, Ambion) treated RNA was electrophoresed in 1% agarose/formaldehyde gels.

    Techniques:

    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Journal: Nature

    Article Title: Induced ncRNAs Allosterically Modify RNA Binding Proteins in cis to Inhibit Transcription

    doi: 10.1038/nature06992

    Figure Lengend Snippet: Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Article Snippet: The soluble DNA-bound RNA fraction was collected after centrifugation at 4,000 g for 15 min. RNA was extracted using Trizol (Invitrogen) and treated with RNase-free DNase I (DNA-free; Ambion).

    Techniques: HAT Assay, Immunoprecipitation, Activity Assay

    α-subunits of RNAP bind to A-boxes upstream of the T-tract. A) DNA sequence of the P sabA upstream region showing the predicted UP-like elements and multiple A-boxes (red boxes). Red, blue and green lines mark the interaction sites of σ 70 -RNAP found by Footprint analysis, correspondingly, see Fig. 5B–C . B–C) Mapping of the binding site for σ 70 -RNAP to P sabA DNA using DNase I footprint assay. 10 nM of [γ 32 P]ATP-labeled P sabA DNA (−166 to +74) were mixed with increasing concentrations of σ 70 -RNAP (0, 6.25, 12.5, 25, or 50 nM). The regions protected from DNase I cleavage are marked by red (core promoter), blue (proximal UP-like element) and green (distal UP-like element) lines. The positions of the T-tract, predicted −35 and −10, and +1 transcriptional start site, are indicated to the left. The stars mark the region of the promoter that was deleted in Δ 46 variants (−97 to −49, see also Fig. S2 and S6A ). Nucleotide positions, relative to the transcriptional start site, are shown to the right. D) Binding of σ 70 -RNAP (55 nM) to P sabA DNA (−166 to +74), with different repeat tract compositions and promoter mutant variants, analyzed by SPR. The sensorgrams show values normalized to that of the full-length T 13 -variant. Binding to a sabA CDS-fragment, also used in Fig. 4 , is shown as a background curve in the top diagram. The bottom diagram is an enlargement of the dotted-lined square in the top diagram. E) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing P sabA with proximal UP-like element deleted. The constructs contain different tract lengths and compositions (see Fig. 5B–C and S6A ). Black bars represent wt promoters and white bars Δ 46 variants, respectively. β-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods . Data is presented as relative values with activity of P sabA T 13 wt set to 1. F) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing sabA promoter with scrambled UP-like elements. β-galactosidase assays were performed as described in Fig. 5E and data is presented as relative values with activity of P sabA wt set to 1.

    Journal: PLoS Pathogens

    Article Title: A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

    doi: 10.1371/journal.ppat.1004234

    Figure Lengend Snippet: α-subunits of RNAP bind to A-boxes upstream of the T-tract. A) DNA sequence of the P sabA upstream region showing the predicted UP-like elements and multiple A-boxes (red boxes). Red, blue and green lines mark the interaction sites of σ 70 -RNAP found by Footprint analysis, correspondingly, see Fig. 5B–C . B–C) Mapping of the binding site for σ 70 -RNAP to P sabA DNA using DNase I footprint assay. 10 nM of [γ 32 P]ATP-labeled P sabA DNA (−166 to +74) were mixed with increasing concentrations of σ 70 -RNAP (0, 6.25, 12.5, 25, or 50 nM). The regions protected from DNase I cleavage are marked by red (core promoter), blue (proximal UP-like element) and green (distal UP-like element) lines. The positions of the T-tract, predicted −35 and −10, and +1 transcriptional start site, are indicated to the left. The stars mark the region of the promoter that was deleted in Δ 46 variants (−97 to −49, see also Fig. S2 and S6A ). Nucleotide positions, relative to the transcriptional start site, are shown to the right. D) Binding of σ 70 -RNAP (55 nM) to P sabA DNA (−166 to +74), with different repeat tract compositions and promoter mutant variants, analyzed by SPR. The sensorgrams show values normalized to that of the full-length T 13 -variant. Binding to a sabA CDS-fragment, also used in Fig. 4 , is shown as a background curve in the top diagram. The bottom diagram is an enlargement of the dotted-lined square in the top diagram. E) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing P sabA with proximal UP-like element deleted. The constructs contain different tract lengths and compositions (see Fig. 5B–C and S6A ). Black bars represent wt promoters and white bars Δ 46 variants, respectively. β-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods . Data is presented as relative values with activity of P sabA T 13 wt set to 1. F) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing sabA promoter with scrambled UP-like elements. β-galactosidase assays were performed as described in Fig. 5E and data is presented as relative values with activity of P sabA wt set to 1.

    Article Snippet: Before cDNA synthesis the total RNA (250 µg/µl) was treated an extra time with Turbo DNase I (Ambion) to remove any residual DNA. cDNA synthesis was performed in 20 µl reactions using 500 ng Turbo DNase treated total RNA, Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science) and random hexamers (60 µM) provided with the kit, according to the manufacturer's protocol. cDNA synthesis was performed at 25°C for 10 min and at 55°C for 30 min.

    Techniques: Sequencing, Binding Assay, Labeling, Mutagenesis, SPR Assay, Variant Assay, Activity Assay, Construct

    Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Binding Assay, Footprinting, Labeling, Sequencing, Purification

    Gene expression analysis of the  mur  genes. (A) Transcription analysis of intergenic region of the selected  mur  genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions.  mur10 ← mur11  means that the detected region between  mur10  and  mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of  mur11  and  mur27  for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Journal: PLoS ONE

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    doi: 10.1371/journal.pone.0076068

    Figure Lengend Snippet: Gene expression analysis of the mur genes. (A) Transcription analysis of intergenic region of the selected mur genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. mur10 ← mur11 means that the detected region between mur10 and mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of mur11 and mur27 for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Article Snippet: For binding site analysis, the reaction mixture contained 500 cps 32 P-lablelled DNA fragments (50 nM), after the binding of protein with DNA, the reaction mixture was incubated in ice bath for 5 min prior to addition of 2.5 µl DNase I buffer and 0.3 U of DNase I (Fermentas), then was carried out for further incubation at 30°C for 1 min.

    Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control, Incubation

    Detection of MDV transcripts in BMMs and BMDCs infected with MDV in vitro . BMMs and BMDCs were infected in vitro with EGFP-expressing MDV. After 3 days, EGFP-positive cells were sorted and RT-PCR was carried out for the detection of (a) immediate early ICP4 (200 bp), (b) early pp38 (198 bp), (c) late gB (193 bp) and (d) MDV-specific l -Meq (200 bp) transcripts. L, ladder; +, positive control MDV-infected CEFs; −, negative control, nuclease-free H 2 O; M, infected BMMs (cDNA); MN, infected BMMs no-RT control (DNase-treated RNA); D, infected BMDCs (cDNA); DN, infected BMDCs no-RT control (DNase-treated RNA).

    Journal: The Journal of General Virology

    Article Title: Marek's disease virus infection of phagocytes: a de novo in vitro infection model

    doi: 10.1099/jgv.0.000763

    Figure Lengend Snippet: Detection of MDV transcripts in BMMs and BMDCs infected with MDV in vitro . BMMs and BMDCs were infected in vitro with EGFP-expressing MDV. After 3 days, EGFP-positive cells were sorted and RT-PCR was carried out for the detection of (a) immediate early ICP4 (200 bp), (b) early pp38 (198 bp), (c) late gB (193 bp) and (d) MDV-specific l -Meq (200 bp) transcripts. L, ladder; +, positive control MDV-infected CEFs; −, negative control, nuclease-free H 2 O; M, infected BMMs (cDNA); MN, infected BMMs no-RT control (DNase-treated RNA); D, infected BMDCs (cDNA); DN, infected BMDCs no-RT control (DNase-treated RNA).

    Article Snippet: RT-PCR RNA samples were extracted using RNeasy Mini Kits (Qiagen) and treated with DNase (Ambion Turbo DNA-free Kits, Life Technologies).

    Techniques: Infection, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control

    Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with DNase I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Curcumin induces Bex genes in N2a neuroblastoma cells in a dose-dependent manner. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated either with 10, 25 or 50 μM of curcumin or with equal amount of DMSO (controls) in serum free media for 2 hours. Total RNA was isolated by Trizol reagent and treated with DNase I to remove any DNA contamination. Reverse transcription on 5 μg of DNA-free RNA was performed and Bex cDNAs were amplified either for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis shows a dose-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S9 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex4 ( d ) and Bex6 ( e ) mRNA amplicons was performed using Image lab software and values obtained were normalized with respective GAPDH band intensity, and were plotted as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Techniques: Cell Culture, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Software, Two Tailed Test

    Curcumin is a specific inducer of all the Bex genes. ( a ) Approximately 80% confluent N2a cells were serum starved for 2 hours and treated with either 2.5, 5, 10 μM of MPTQ or 25 μM of curcumin in serum free DMEM for 4 hours. Equivalent amount of DMSO treated N2a cells were considered as controls. Total RNA was isolated, DNase I treated and expression of Bex mRNAs was studied by RT-PCR analysis. GAPDH was used as loading control. The original gel images are shown in Supplementary Fig. S11 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products was performed and normalized with corresponding GAPDH band intensity. Values are displayed as histograms of mean ± standard deviation from three independent experiments, which indicates induction of all endogenous Bex genes are observed in curcumin but not in MPTQ treated N2a cells. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Curcumin is a specific inducer of all the Bex genes. ( a ) Approximately 80% confluent N2a cells were serum starved for 2 hours and treated with either 2.5, 5, 10 μM of MPTQ or 25 μM of curcumin in serum free DMEM for 4 hours. Equivalent amount of DMSO treated N2a cells were considered as controls. Total RNA was isolated, DNase I treated and expression of Bex mRNAs was studied by RT-PCR analysis. GAPDH was used as loading control. The original gel images are shown in Supplementary Fig. S11 . Densitometric analysis of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products was performed and normalized with corresponding GAPDH band intensity. Values are displayed as histograms of mean ± standard deviation from three independent experiments, which indicates induction of all endogenous Bex genes are observed in curcumin but not in MPTQ treated N2a cells. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Techniques: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation, Two Tailed Test

    Optimization of curcumin treatment duration for the induction of Bex genes in N2a cells. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated with 25 μM of curcumin for 2, 4, 8 or 24 hours. Control cells were treated with equivalent amount of DMSO for 2 hours. Total RNA was isolated at indicated time points using Trizol reagent and treated with DNase I for 15 minutes at room temperature. RT-PCR analysis of DNA-free Bex mRNA was performed for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis clearly demonstrates a time-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S10 . Intensity of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products were obtained and normalized with corresponding GAPDH band intensity, and displayed as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Journal: Scientific Reports

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    doi: 10.1038/srep41420

    Figure Lengend Snippet: Optimization of curcumin treatment duration for the induction of Bex genes in N2a cells. ( a ) N2a cells (3 × 10 5 cells) were cultured for two days in 25 cm 2 flask, serum starved for 2 hours and treated with 25 μM of curcumin for 2, 4, 8 or 24 hours. Control cells were treated with equivalent amount of DMSO for 2 hours. Total RNA was isolated at indicated time points using Trizol reagent and treated with DNase I for 15 minutes at room temperature. RT-PCR analysis of DNA-free Bex mRNA was performed for 32, 34 or 36 PCR cycles. Agarose gel electrophoresis clearly demonstrates a time-dependent induction of Bex genes by curcumin. The original gel images are shown in Supplementary Fig. S10 . Intensity of Bex1 ( b ), Bex2 ( c ), Bex3 ( d ), Bex4 ( e ) and Bex6 ( f ) PCR products were obtained and normalized with corresponding GAPDH band intensity, and displayed as histograms of mean ± standard error of mean from three independent experiments. P-values displayed were calculated by using two-tailed, unpaired Student’s t-test and * = p ≤ 0.05 is considered statistically significant.

    Article Snippet: Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Techniques: Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Two Tailed Test

    Overview of RT-RamDA and single-cell RamDA-seq.  a  Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I.  b  Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog ,  Pou5f1 ,  Zfp42 , and  Sox2 ) and three housekeeping ( Gnb2l1 ,  Atp5a1 , and  Tubb5 ) genes using a conventional method (−) as a standard.  c  Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section.  d  Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in  b  and  d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile.  e  Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in  d  and  e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: A mixture containing 2 μL of conventional RT mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18 (Thermo Fisher), 8 pmol random hexamers (TaKaRa), and 1.5× PrimeScript enzyme mix in RNase-free water) or 2 μL of RT-RamDA mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18, 8 pmol random hexamers or NSRs, 0.2 U of DNase I Amplification Grade (Thermo Fisher), 100 ng of T4 gene 32 protein (Roche), and 1.5× PrimeScript enzyme mix in RNase-free water) was added to 1 μL of diluted, denatured template RNA.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    Primer-template dependent inhibitory effect on mRNA amplification . RNA samples extracted from cells harvested at 2,1 mg (dry weight)/mL were treated with DNase I according to protocol II. Different set of primers (as described in table I) was used to amplify CNA2 (lane 2), ACT1 (lane 3), TPS2 (lane 4), PDA1 (lane 5), CNA1 (lane 6), TPS1 (lane 7) mRNAs. 100 bp DNA ladder (lane P) and RT-minus control using ACT1 primers (lane 1).

    Journal: BMC Molecular Biology

    Article Title: Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

    doi: 10.1186/1471-2199-6-9

    Figure Lengend Snippet: Primer-template dependent inhibitory effect on mRNA amplification . RNA samples extracted from cells harvested at 2,1 mg (dry weight)/mL were treated with DNase I according to protocol II. Different set of primers (as described in table I) was used to amplify CNA2 (lane 2), ACT1 (lane 3), TPS2 (lane 4), PDA1 (lane 5), CNA1 (lane 6), TPS1 (lane 7) mRNAs. 100 bp DNA ladder (lane P) and RT-minus control using ACT1 primers (lane 1).

    Article Snippet: Protocol II was performed using the DNase I amplification grade kit (Life Technologies, Inc.) following the recommended procedure.

    Techniques: Amplification

    Effect of EDTA concentration on CNA1 mRNA amplification by RT-PCR . RNA samples extracted from cells harvested at 1.2 mg (dry weight/mL) were treated with DNase I according to protocol II. The reactions were stopped by the addition of 1.25 mM (lane 1), 1.5 mM (lane 2), 1.75 mM (lane 3); 2.0 mM (lane 4) and 2.25 mM (lane 5) of EDTA and treated samples were amplified by RT-PCR using CNA1 primers. P, 100 bp DNA Ladder (BioLabs-Inc).

    Journal: BMC Molecular Biology

    Article Title: Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR

    doi: 10.1186/1471-2199-6-9

    Figure Lengend Snippet: Effect of EDTA concentration on CNA1 mRNA amplification by RT-PCR . RNA samples extracted from cells harvested at 1.2 mg (dry weight/mL) were treated with DNase I according to protocol II. The reactions were stopped by the addition of 1.25 mM (lane 1), 1.5 mM (lane 2), 1.75 mM (lane 3); 2.0 mM (lane 4) and 2.25 mM (lane 5) of EDTA and treated samples were amplified by RT-PCR using CNA1 primers. P, 100 bp DNA Ladder (BioLabs-Inc).

    Article Snippet: Protocol II was performed using the DNase I amplification grade kit (Life Technologies, Inc.) following the recommended procedure.

    Techniques: Concentration Assay, Amplification, Reverse Transcription Polymerase Chain Reaction