RNA was isolated using TRIzol® Reagent (Invitrogen) according to the manufacturer’s protocol and reverse transcribed into cDNA using qScript™ Reagent (Quanta Biosciences). mRNA levels were measured as previously described30 (link). Primer sequences are listed in Supplementary Table 1 .
Qscript reagent
Manufactured by Quanta Biosciences
The QScript™ Reagent is a reverse transcription reagent designed for the synthesis of complementary DNA (cDNA) from RNA templates. It includes a thermostable reverse transcriptase enzyme, necessary buffers, and other components required for the reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
7500 software v2, by Thermo Fisher Scientific (1 mentions)
Sybr green real time pcr, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Perfecta sybr green fastmix reagent, by Quanta Biosciences (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
4 protocols using qscript reagent
1
RNA Isolation and mRNA Quantification
2
Quantitative Real-Time PCR Transcriptomics
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Total RNA was isolated using TRIzol® Reagent (Life Technologies) according to the manufacturer’s instructions and reverse transcribed using qScript™ Reagent (Quanta Biosciences). Analysis of mRNA levels was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) with SYBR® Green Real-Time PCR, using primers designed using the NIH primer design tool. The ribosomal housekeeping gene L32 was used as an internal control and data was analyzed with the 7500 Software v2.0.5 (Applied Biosystems). Student’s t-test was performed for statistical significance (see Supplementary Table 1 for qPCR primer sequences).
3
RNA Extraction and RT-qPCR Analysis
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RNA was collected from corresponding B-cell samples and reverse transcribed using the qScript reagent (Quanta Biosciences). RT-qPCR was performed with the StepOne Plus Real-Time PCR System (Applied Biosystems) using PerfeCTa SYBR Green FastMix reagent (Quanta Biosciences) or TaqMan MicroRNA Assay (Life Technologies). Primer sequences used are listed in Table S2 in Supplementary Material.
4
RNA Isolation and RT-qPCR Analysis
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RNA was isolated using TRizol Reagent (Life Technologies; catalog no. 15596026) according to the manufacturer's instructions. Reverse transcription-qPCR (RT-qPCR) was performed on 2 ng/μL of isolated RNA using qScript Reagent (Quanta Biosciences; catalog no. 76047–074). The mRNA levels were analyzed on a 7500 Fast Real-Time PCR System (Applied Biosystems; catalog no. 4359284) with SYBR Green Real-Time PCR. Primers were designed using the NIH primer design tool. mRNA expression was normalized to the internal control ribosomal housekeeping gene L32. RT-qPCR was performed with primers listed in Supplementary Table S4.
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