Protoscript 2 reverse transcription

Manufactured by New England Biolabs

Sourced in Canada

ProtoScript II is a reverse transcriptase enzyme used for the conversion of RNA to complementary DNA (cDNA). It exhibits high thermostability and can be used for a variety of RNA templates, including mRNA, total RNA, and viral RNA.

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Lab products found in correlation

Megascript rnai kit, by Thermo Fisher Scientific (2 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions) Nanodrop technology, by Thermo Fisher Scientific (1 mentions) Ez 10 spin column total rna miniprep kit, by Bio Basic (1 mentions) Lightcycler 480 system, by Roche (1 mentions) Quantitect reverse transcription kit, by Qiagen (1 mentions) Cfx96 real time pcr detection system, by Bio-Rad (1 mentions) Experion rna analysis kit, by Bio-Rad (1 mentions) Cfx manager, by Bio-Rad (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions)

Spelling variants of this product

ProtoScript II (94 citations) ProtoScript II Reverse Transcription kit (5 citations)

4 protocols using protoscript 2 reverse transcription

Total RNA Extraction and qPCR Analysis

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Cells were collected by trypsinization and centrifugation, then washed once with 10% BSA in PBS and once with PBS. RNA was prepared using the EZ-10 Spin Column Total RNA Miniprep Kit [BS1361(SK8655); BioBasic] according to the manufacturer’s instructions. RNA was quantified using NanoDrop technology (Thermo Fisher Scientific), and, for each sample, 1 µg RNA was converted to cDNA using ProtoScript II reverse transcription (M0368; New England Biolabs) in a 20-μl reaction. 0.5 μl of reverse transcription was used per 10-μl qPCR (Luna Universal qPCR Master Mix).
Total RNA from mouse muscle was extracted using TRIzol and then transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Expression of selected genes was analyzed using the LightCycler 480 system (Roche) and SYBR Green chemistry. All qPCR results were presented relative to the mean of 36b4, B2m, and/or Gapdh (comparative cycle threshold method). Primer sequences are specified in Table 1.

Addicks G.C., Zhang H., Ryu D., Vasam G., Green A.E., Marshall P.L., Patel S., Kang B.E., Kim D., Katsyuba E., Williams E.G., Renaud J.M., Auwerx J, & Menzies K.J. (2022). GCN5 maintains muscle integrity by acetylating YY1 to promote dystrophin expression. The Journal of Cell Biology, 221(2), e202104022.

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Quantitative PCR Analysis of mRNA Expression

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Relative expression levels of mRNA were determined by real-time quantitative PCR as described previously (French et al., 2015 (link)). In brief, total RNA was extracted from 14–20 retinas from each experimental group, using a RNeasy Plus mini kit (Qiagen). mRNA was evaluated using an Experion RNA Analysis kit (Bio-Rad). 50 ng total RNA was used for first-strand cDNA synthesis with ProtoScript II reverse transcription (New England BioLabs). Quantitative PCR was performed using GoTaq qPCR Master (Promega) on a CFX96TM real-time PCR detection system (Bio-Rad). All PCR runs were performed three times. Gene expression levels, PCR efficiency, and the standard error of measurement were calculated using CFX Manager (Bio-Rad). Primer sequences for the specific and reference genes are provided elsewhere (French et al., 2015 (link)). Amplification efficiencies of the primers were determined using serially diluted cDNA samples.

Frolov R.V., Immonen E.V., Saari P., Torkkeli P.H., Liu H, & French A.S. (2018). Phenotypic plasticity in Periplaneta americana photoreceptors. The Journal of General Physiology, 150(10), 1386-1396.

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Transcriptome-based Cockroach Gqα and PLC RNAi

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The putative P. americana Gqα and phosphoinositide-specific PLC were identified from the retinal transcriptome as described for other cockroach genes previously in detail (French 2012 (link); French et al. 2015 (link)). Long double-stranded RNA (540 bp for Gqα and 683 bp for PLC) was synthesized using similar methods as described earlier (French et al. 2015 (link)). Reverse transcription was performed using total RNA extracted from cockroach retinas and oligo-d(T)23VN primers with ProtoScript II reverse transcription (New England Biolabs, Whitby, Ontario, Canada). The reverse transcription product was used in PCRs to amplify the template DNAs using Q5 High-Fidelity DNA Polymerase (New England Biolabs). dsRNA was synthesized with the MEGAscript RNAi kit (Thermo Fisher Scientific, Waltham MA). Cockroaches were anaesthetised with CO₂ and Hamilton 5-µL syringe with a beveled needle attached was used to inject 1 µL of the dsRNA (4 µg/µL injection buffer that contained 0.1 µM Na phosphate buffer and 5 µM KCl) into the head. Control animals were injected with 1 µL of the injection buffer. 8–10 cockroaches were used for each gene and for the control. After the injections, animals were maintained in separate cages but otherwise under identical conditions to normal.

Ignatova I.I., French A.S., Torkkeli P.H., Liu H, & Frolov R.V. (2020). Suppression of Gq and PLC gene expression has a small effect on quantum bumps in vivo in Periplaneta americana. Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology, 206(4), 597-610.

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dsRNA Injection in Cockroach Retina

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dsRNA was synthesized and injected (4–5 µg in 1 µl Ringer solution) into the head tissue under CO₂ anesthesia as described previously (French et al., 2015 (link); Immonen et al., 2017 (link)). In brief, reverse transcription was performed using total RNA extracted from cockroach retinas and oligo d(T)₂₃VN primers with ProtoScript II reverse transcription (New England Biolabs, Inc.). The reverse transcription product was used in PCRs to amplify the template DNAs using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Inc.). dsRNA was synthesized with the MEGAscript RNAi kit (Ambion, Thermo Fisher Scientific). The GenBank accession numbers for P. americana TRP and TRPL sequences are KC329816 and KC292630, respectively. The primers and complete dsRNA sequences have been published before (French et al., 2015 (link); Immonen et al., 2017 (link)). For injection, a small hole was made in the in chitin of the frontal part of the head below an imaginary line connecting the antennas. Solution was delivered using a sterile disposable glass pipette. After the injection, animals were maintained in separate cages at 25°C. Control animals either received no injection or were injected with 1 µl cockroach Ringer solution.

Saari P., French A.S., Torkkeli P.H., Liu H., Immonen E.V, & Frolov R.V. (2017). Distinct roles of light-activated channels TRP and TRPL in photoreceptors of Periplaneta americana. The Journal of General Physiology, 149(4), 455-464.

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