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    Thermo Fisher superscript iii reverse transcriptase
    Accumulation ( A ) and silencing activity ( B ) of cholesterol-modified siRNAs and TsiRNAs in a carrier-free mode in vitro. ( A ) Accumulation of monomer (Ch-siRNA) and trimeric (Ch-TsiRNA-2) cholesterol-modified siRNAs in KB-8-5 cells 4 h after addition (1 µM), measured by stem-loop <t>RT-PCR.</t> B) Silencing activity of Ch-siRNA, Ch-TsiRNA-1 and Ch-TsiRNA-2 <t>three</t> days after addition (5 µM) to KB-8-5-MDR1-GFP or KB-3-1-MDR1-GFP cells, fluorescence intensity value of untreated cells was used as a 100%.
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 90863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Accumulation ( A ) and silencing activity ( B ) of cholesterol-modified siRNAs and TsiRNAs in a carrier-free mode in vitro. ( A ) Accumulation of monomer (Ch-siRNA) and trimeric (Ch-TsiRNA-2) cholesterol-modified siRNAs in KB-8-5 cells 4 h after addition (1 µM), measured by stem-loop RT-PCR. B) Silencing activity of Ch-siRNA, Ch-TsiRNA-1 and Ch-TsiRNA-2 three days after addition (5 µM) to KB-8-5-MDR1-GFP or KB-3-1-MDR1-GFP cells, fluorescence intensity value of untreated cells was used as a 100%.

    Journal: Molecules

    Article Title: Trimeric Small Interfering RNAs and Their Cholesterol-Containing Conjugates Exhibit Improved Accumulation in Tumors, but Dramatically Reduced Silencing Activity

    doi: 10.3390/molecules25081877

    Figure Lengend Snippet: Accumulation ( A ) and silencing activity ( B ) of cholesterol-modified siRNAs and TsiRNAs in a carrier-free mode in vitro. ( A ) Accumulation of monomer (Ch-siRNA) and trimeric (Ch-TsiRNA-2) cholesterol-modified siRNAs in KB-8-5 cells 4 h after addition (1 µM), measured by stem-loop RT-PCR. B) Silencing activity of Ch-siRNA, Ch-TsiRNA-1 and Ch-TsiRNA-2 three days after addition (5 µM) to KB-8-5-MDR1-GFP or KB-3-1-MDR1-GFP cells, fluorescence intensity value of untreated cells was used as a 100%.

    Article Snippet: Synthesis of cDNA and stem-loop PCR was carried out using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA); and qPCR mix was assembled using BioMaster qPCR SYBR Blue (Biolabmix, Novosibirsk, Russia).

    Techniques: Activity Assay, Modification, In Vitro, Reverse Transcription Polymerase Chain Reaction, Fluorescence

    Detection of 5′ trans-splicing in vitro . (a) Left: Anti-Flag Western Blot on total proteins from 3T3 cells transfected with 5′-PTM or a plasmid expressing full length Dnm2-Flag fusion protein as control (Dynaflag). NT: nontransfected. Right: Dnm2 immunoprecipitation followed by Flag western blot on 3T3 cells transfected with 5′-PTM. The image is overexposed and over contrasted to enhance the detection of the band indicated by an asterisk. (b) RT-PCR analysis on mRNA extracted from 3T3 cells transfected with 5′-PTM. Dyna-endo: endogenous Dnm2 transcripts amplified using E10EndoF/E12endoR primers, TS: trans-spliced RNA amplified using E8OptiF/Ex17endoR and Ex11OptiF/Ex14endoR primers; PTM are amplified using E8OptF/Ex11OptiR primers. C−: negative PCR control. The isoform of Dnm2 identified after sequencing for each band is indicated below ( i.e. , isoform 2 or 4 or both). 3T3 cells were transfected three times. (c) Top: Schematic representation of Dnm2 alternative splicing leading to isoforms 2 and 4, locations of primers used for PCR amplification are depicted. Bottom: Example of heterozygous trans-spliced sequence showing Ex13 optimized sequence followed by ex13 bis and ex14 endogenous sequence. The orange squares show the optimized nucleotides. (d) Anti-Flag and anti-DNM2 immunofluorescence on 3T3 cells 48 hours after transfection with AS2-, AS3-, noAS-▵pA-5′-PTM and full length Dnm2-Flag fusion constructs (scale bar = 10 µm). PTM, pre-trans-splicing molecules; RT-PCR, reverse transcription-polymerase chain reaction.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    doi: 10.1038/mtna.2016.67

    Figure Lengend Snippet: Detection of 5′ trans-splicing in vitro . (a) Left: Anti-Flag Western Blot on total proteins from 3T3 cells transfected with 5′-PTM or a plasmid expressing full length Dnm2-Flag fusion protein as control (Dynaflag). NT: nontransfected. Right: Dnm2 immunoprecipitation followed by Flag western blot on 3T3 cells transfected with 5′-PTM. The image is overexposed and over contrasted to enhance the detection of the band indicated by an asterisk. (b) RT-PCR analysis on mRNA extracted from 3T3 cells transfected with 5′-PTM. Dyna-endo: endogenous Dnm2 transcripts amplified using E10EndoF/E12endoR primers, TS: trans-spliced RNA amplified using E8OptiF/Ex17endoR and Ex11OptiF/Ex14endoR primers; PTM are amplified using E8OptF/Ex11OptiR primers. C−: negative PCR control. The isoform of Dnm2 identified after sequencing for each band is indicated below ( i.e. , isoform 2 or 4 or both). 3T3 cells were transfected three times. (c) Top: Schematic representation of Dnm2 alternative splicing leading to isoforms 2 and 4, locations of primers used for PCR amplification are depicted. Bottom: Example of heterozygous trans-spliced sequence showing Ex13 optimized sequence followed by ex13 bis and ex14 endogenous sequence. The orange squares show the optimized nucleotides. (d) Anti-Flag and anti-DNM2 immunofluorescence on 3T3 cells 48 hours after transfection with AS2-, AS3-, noAS-▵pA-5′-PTM and full length Dnm2-Flag fusion constructs (scale bar = 10 µm). PTM, pre-trans-splicing molecules; RT-PCR, reverse transcription-polymerase chain reaction.

    Article Snippet: Total RNA (1 µg) was submitted to reverse transcription using the Superscript III reverse transcriptase kit (Life Technologies) using random primers. cDNA were amplified by PCR under the following conditions: 96°C for 5 minutes, cycles of 30 seconds at 96°C, 30 seconds at the appropriate temperature (58 to 61°C), 30 seconds to 1 minute at 72°C, and a final step of 7 minutes at 72°C.

    Techniques: In Vitro, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Sequencing, Immunofluorescence, Construct

    Induction of Id1-3 ( A ), endogenous expression of p21 and p53 ( B ), and expression of p53 ( C ), p21 ( D ), p27 ( E ), p107 ( F ) and p130 ( G ) upon recombinant BMP4 administration to cell cultures 24hrs after administration of 40ng/ml recombinant BMP4. Messenger RNA expression levels of Id1, Id2 and Id3 are significantly up-regulated in WERI-Rb-1 ( A ) upon a BMP4 administration, whereas no changes in p53, p21, p27, p107 and p130 expression level were detectable ( C-G ). Endogeneous levels of p21 and p53 are, however, elevated in WERI-Rb1 cells ( B ). Data were gained from real-time PCR analyses of three independent experiments. Cells that were only treated with 0.1% BSA in 4mM HCl, the solvent for recombinant human BMP4, were taken as a control (ctr.) group for A and C-G , healthy human retina was used as a reference in B and the expression level was set as 1, respectively.

    Journal: International Journal of Biological Sciences

    Article Title: Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells

    doi:

    Figure Lengend Snippet: Induction of Id1-3 ( A ), endogenous expression of p21 and p53 ( B ), and expression of p53 ( C ), p21 ( D ), p27 ( E ), p107 ( F ) and p130 ( G ) upon recombinant BMP4 administration to cell cultures 24hrs after administration of 40ng/ml recombinant BMP4. Messenger RNA expression levels of Id1, Id2 and Id3 are significantly up-regulated in WERI-Rb-1 ( A ) upon a BMP4 administration, whereas no changes in p53, p21, p27, p107 and p130 expression level were detectable ( C-G ). Endogeneous levels of p21 and p53 are, however, elevated in WERI-Rb1 cells ( B ). Data were gained from real-time PCR analyses of three independent experiments. Cells that were only treated with 0.1% BSA in 4mM HCl, the solvent for recombinant human BMP4, were taken as a control (ctr.) group for A and C-G , healthy human retina was used as a reference in B and the expression level was set as 1, respectively.

    Article Snippet: Equal amounts of RNA were subjected to an RT-reaction using oligo(dt)20 primers (Invitrogen), employing the SuperScript III reverse transcriptase system (Invitrogen) and following the manufacturer`s instructions.

    Techniques: Expressing, Recombinant, RNA Expression, Real-time Polymerase Chain Reaction

    RNA editing of EEF-derived miRNA. (A) Graphic illustration of A-to-I editing, which is catalyzed by ADAR enzymes. miRNA editing can occur in seed regions (as shown here) or outside of the seed regions. This graph was created with BioRender. (B) Boxplots showing the editing levels (median with interquartile range), in control and KA samples at the two different time points for six edited sites detected in EEF miRNAs. (C) The table shows evidence supporting the edited sites and (D) sequence motifs of the edited sites. This shows the nucleotide preferences of all sequences surrounding the six edited sites [three-bases long, with the edited nucleotide Adenosine (A) is centered]. All the six edited sites have preferable sequence motifs for ADAR enzymes, which is (1) over-represented uridine (U) but depleted guanosine (G) at the nucleotides before the edited sites and (2) over-represented guanosine at the nucleotides after the edited sites. Confidence of edited site ( Li et al., 2018 ) reported in brain ( Alon et al., 2012 ) and reported in exosomes ( Nigita et al., 2018 ).

    Journal: Frontiers in Neuroscience

    Article Title: Altered Biogenesis and MicroRNA Content of Hippocampal Exosomes Following Experimental Status Epilepticus

    doi: 10.3389/fnins.2019.01404

    Figure Lengend Snippet: RNA editing of EEF-derived miRNA. (A) Graphic illustration of A-to-I editing, which is catalyzed by ADAR enzymes. miRNA editing can occur in seed regions (as shown here) or outside of the seed regions. This graph was created with BioRender. (B) Boxplots showing the editing levels (median with interquartile range), in control and KA samples at the two different time points for six edited sites detected in EEF miRNAs. (C) The table shows evidence supporting the edited sites and (D) sequence motifs of the edited sites. This shows the nucleotide preferences of all sequences surrounding the six edited sites [three-bases long, with the edited nucleotide Adenosine (A) is centered]. All the six edited sites have preferable sequence motifs for ADAR enzymes, which is (1) over-represented uridine (U) but depleted guanosine (G) at the nucleotides before the edited sites and (2) over-represented guanosine at the nucleotides after the edited sites. Confidence of edited site ( Li et al., 2018 ) reported in brain ( Alon et al., 2012 ) and reported in exosomes ( Nigita et al., 2018 ).

    Article Snippet: For analysis of transcripts, complementary DNA (cDNA) was produced from 1 μg of the total RNA by reverse transcription using Superscript III Reverse Transcriptase enzyme (Invitrogen).

    Techniques: Derivative Assay, Sequencing

    SpxA1DD and SpxA2DD activate several Bacillus anthracis genes in B. subtilis . Promoters of selected genes (horizontal axis) controlled by either SpxA1DD or SpxA2DD, as identified by microarray, were transcriptionally fused to a promoterless lacZ and integrated into the chromosome at the thrC locus of a B. subtilis JH642 Δ spx strain harboring an IPTG-inducible copy of either spxA1DD or spxA2DD integrated at the amyE locus. β-galactos idase activi ty was measured in these strains at 30 min. intervals after the addition of 1 mmol/L IPTG. The maximal activity, which was usually observed at 1–2 h after IPTG induction, was divided by the β-galactosidase activity when IPTG was added; except for P spxA1 -lacZ , where the minimal activity was divided by the β-galactosidase activity when IPTG was added (SpxA1DD: minus IPTG 0.64 ± 0.16, plus IPTG 0.49 ± 0.2; Sp xA2 DD : minus IPTG 0.52 ± 0.27, plus IPTG 0.44 ± 0.18). The ratio was shown as the average of three biological triplicates with standard deviation. Significance was determined by a two-tailed T -test comparing plus IPTG to minus IPTG ratios; * P

    Journal: MicrobiologyOpen

    Article Title: Transcriptomic and phenotypic analysis of paralogous spx gene function in Bacillus anthracis Sterne

    doi: 10.1002/mbo3.109

    Figure Lengend Snippet: SpxA1DD and SpxA2DD activate several Bacillus anthracis genes in B. subtilis . Promoters of selected genes (horizontal axis) controlled by either SpxA1DD or SpxA2DD, as identified by microarray, were transcriptionally fused to a promoterless lacZ and integrated into the chromosome at the thrC locus of a B. subtilis JH642 Δ spx strain harboring an IPTG-inducible copy of either spxA1DD or spxA2DD integrated at the amyE locus. β-galactos idase activi ty was measured in these strains at 30 min. intervals after the addition of 1 mmol/L IPTG. The maximal activity, which was usually observed at 1–2 h after IPTG induction, was divided by the β-galactosidase activity when IPTG was added; except for P spxA1 -lacZ , where the minimal activity was divided by the β-galactosidase activity when IPTG was added (SpxA1DD: minus IPTG 0.64 ± 0.16, plus IPTG 0.49 ± 0.2; Sp xA2 DD : minus IPTG 0.52 ± 0.27, plus IPTG 0.44 ± 0.18). The ratio was shown as the average of three biological triplicates with standard deviation. Significance was determined by a two-tailed T -test comparing plus IPTG to minus IPTG ratios; * P

    Article Snippet: Microarray design, data collection, and data analysis cDNA for microarray experiments were generated by adding 2 μg of total RNA in a mixture containing 6 μg of random hexamers (Life Technologies, Carlsbad, CA), 0.01 mol/L dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mmol/L each dATP, dCTP, and dGTP, 15 mmol/L dTTP, and 10 mmol/L amino-allyl-dUTP (aa-dUTP) (Sigma), reaction buffer, and 400 units of SuperScript III reverse transcriptase (RT) (Life Technologies) at 42°C overnight.

    Techniques: Microarray, Activity Assay, Standard Deviation, Two Tailed Test

    Viral loads and plasma levels of IFNα, IL-15, IL-18 and IL-1βin sample time courses from three subjects acutely infected with HIV. A is a US plasma donor, whose sample time course is plotted in days and is aligned relative to the time (designated day 0) when the plasma viral load first reached 100 copies/ml (i.e. the start of the viral expansion phase). B and C are CHAVI 001 subjects, whose sample time courses are plotted in weeks, and are aligned relative to the time of study enrollment (week 0). Subject B started ART just after study enrollment, as indicated by the black arrow. Viral load data is plotted as open squares joined by dotted lines, and is expressed as log 10 RNA copies/ml. Data for each cytokine is plotted as filled symbols joined by solid lines, and is expressed as pg/ml.

    Journal: PLoS ONE

    Article Title: Cross-Sectional Detection of Acute HIV Infection: Timing of Transmission, Inflammation and Antiretroviral Therapy

    doi: 10.1371/journal.pone.0019617

    Figure Lengend Snippet: Viral loads and plasma levels of IFNα, IL-15, IL-18 and IL-1βin sample time courses from three subjects acutely infected with HIV. A is a US plasma donor, whose sample time course is plotted in days and is aligned relative to the time (designated day 0) when the plasma viral load first reached 100 copies/ml (i.e. the start of the viral expansion phase). B and C are CHAVI 001 subjects, whose sample time courses are plotted in weeks, and are aligned relative to the time of study enrollment (week 0). Subject B started ART just after study enrollment, as indicated by the black arrow. Viral load data is plotted as open squares joined by dotted lines, and is expressed as log 10 RNA copies/ml. Data for each cytokine is plotted as filled symbols joined by solid lines, and is expressed as pg/ml.

    Article Snippet: HIV-1 RNA was reverse transcribed to cDNA using Superscript III Reverse Transcriptase (RT) System (Invitrogen.)

    Techniques: Infection