mRNA expression was quantitated using RT-PCR as previously described [5 (link),46 (link),47 (link),48 (link)]. Briefly, TRIzol (Life Technologies) extracted total RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). Total RNA (250 ng) was subjected to cDNA synthesis using the High Efficiency cDNA Synthesis Kit (Life Technologies). The levels of various mRNA transcripts were determined using Fast SYBR Green (Life Technologies) and gene-specific forward and reverse primer sets (Table 1 ) on a ViiA7 instrument (Life Technologies). 18s RNA expression was used to normalize mRNA expression using the ΔΔCT comparative method.
High efficiency cdna synthesis kit
Manufactured by Thermo Fisher Scientific
The High Efficiency cDNA Synthesis Kit is a laboratory tool designed for the efficient conversion of RNA into complementary DNA (cDNA). The kit provides the necessary reagents and protocols to perform this fundamental step in various molecular biology and genomics applications.
Automatically generated - may contain errors
Lab products found in correlation
Viia7 instrument, by Thermo Fisher Scientific (1 mentions)
Fast sybr green, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
2 protocols using high efficiency cdna synthesis kit
1
Quantification of mRNA Expression via RT-PCR
2
Quantitative RT-PCR for Gene Expression Analysis
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Gene expression at the mRNA level was determined by performing quantitative RT-PCR (qRT-PCR) as described earlier [34 (link)]. Briefly, RNA was isolated from tumor tissue and cells using Trizol reagent and prepared cDNA using the high efficiency cDNA synthesis kit (Life Technologies). Gene specific primers were utilized to determine expression levels of genes with SYBR Green PCR Master Mix (Life Technologies) with CFX96 Touch Real-Time PCR Detection system (Bio-Rad). β-actin was used as an internal control. Normalized fold expression of genes was determined using the CFX Manager Software.
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