Cells were lysed with Trizol (Life Technologies) and mRNA extracted using the Direct-zol RNA MiniPrep kit (Zymo, Irvine, CA). mRNA concentration and purity were assessed using a DeNovix DS-11 spectrophotometer and reverse transcription performed using the Superscript III first strand synthesis reverse transcription kit (Invitrogen, Carlsbad, CA) per the manufacturer’s guidelines. Reverse transcription and quantitative PCR was performed using TaqMan Master Mix and Gene Expression Probes (Applied Biosystems, Foster City, CA) as detailed in the supplementary information. Samples were run on the 7500 Fast Real-Time PCR System (Applied Biosystems). Data was analyzed with the ExpressionSuite software using the ∆∆CT method with UBC as the housekeeping gene and untreated or unstimulated T cells for the reference samples.
Superscript 3 first strand synthesis reverse transcription kit
Manufactured by Thermo Fisher Scientific
The Superscript III first strand synthesis reverse transcription kit is a tool used to convert RNA into complementary DNA (cDNA) in a laboratory setting. The kit contains the necessary components, including the Superscript III reverse transcriptase enzyme, to perform this conversion process.
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Lab products found in correlation
4 protocols using superscript 3 first strand synthesis reverse transcription kit
1
Quantifying Gene Expression in T Cells
2
Quantifying BCL-2 Family Gene Expression
3
RNA Extraction and qPCR Analysis
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Total RNA from cell culture was extracted and purified using RNeasy mini kit (Qiagen) according to manufacturer’s instructions. Complementary DNA synthesis was performed with Superscript III reverse transcription First-Strand synthesis kit (Invitrogen). qPCR was performed for each sample in duplicate and gene expression was normalized with the housekeeping gene (HPRT) and relative expression calculated using the ΔΔCt method. Primer sets were synthesized by Integrated DNA Technologies, Inc (See Table S1 for primer sequences).
4
Endothelial RNA Isolation and qRT-PCR
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Endothelial RNA-enriched fractions from mouse aortae were prepared as described previously (Briot et al., 2014 (link)). Total RNA from HAECs was purified using the RNeasy Mini kit (QIAGEN). Complementary DNA synthesis was performed with Superscript III reverse transcription First-Strand synthesis kit (Invitrogen) using oligo-dT primers.
qRT-PCR was performed using primers designed for human targets, provided inTable S1 . Primers for mouse targets have been described previously (Briot et al., 2014 (link)). Each reaction was run in duplicate and normalized with HPRT housekeeping gene. P-values were calculated using the appropriate Student’s t test. Data are represented as mean ± SEM. For gene microarray analysis, only RNA samples with RNA integrity numbers (RIN) of 7.0 or higher were used for subsequent processing.
qRT-PCR was performed using primers designed for human targets, provided in
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