PCR and qRT-PCR analysis of various glyco-genes
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Selection of the proper cDNA synthesis kit is critical for detailed analysis of quantitative gene expression. In order to achieve this goal, a good reverse transcriptase enzyme performance at elevated temperatures is important for efficient reverse transcription of RNA and for specificity when using gene-specific qRT-PCR primers spanning short segment of genes. The Verso enzyme cDNA synthesis kit is widely used for cDNA synthesis, due to its proven ability to produce high yields of fully representative full length cDNA from low yields of RNA.
PCR and qRT-PCR analysis of gene expression.
RNA prepared from cell lysates after Trizol extraction (TRI Reagent® Cat# TR 118, from Molecular Research Center, Inc.)
To synthesize cDNA, 1 µg of RNA was used in 11 µl of molecular biology grade water. RNA was pre-heated at 70°C for 5 min followed by cooling on ice. To this, 9 µl of master mix* was added and cDNA was synthesized at 42°C for 1 hour followed by 92°C for 2 mins. Finally, cDNA was stored at -20 °C until further use. (*20 µL master mix = 4 µL 5X cDNA synthesis buffer + 2 µL 1X dNTP Mix + 1 µL Random Hexamer + 1 µL RT Enhancer + 1 µL Verso Enzyme Mix)
We did not observe any major difference in the expression of various genes either in the presence or absence of anchored oligo-dT during qRT-PCR analysis. If needed, it has to be added in the master mix and reaction volume is adjusted to 20 µl with mol bio grade water.
Using this cDNA synthesis kit, we have validated microarray data and analyzed various gene expressions before and after different drug treatments by PCR as well as qRT-PCR using SYBR® Premix Ex TaqTM (Takara Bio) on a Mastercycler Epgradient realplex (Eppendorf AG).
The cDNA synthesized by this kit was stable at least for 4 years.