When designing a RT-qPCR assay it is important to decide whether to use total RNA or purified mRNA as the template for reverse transcription. mRNA may provide slightly more sensitivity, but total RNA is often used because it has important advantages over mRNA as a starting material.
Three different approaches can be used for priming cDNA reactions in two-step assays: oligo(dT) primers, random primers, or sequence specific primers . Often, a mixture of oligo(dT)s and random primers is used. These primers anneal to the template mRNA strand and provide reverse transcriptase enzymes a starting point for synthesis.
Alloysius Chibuike Ogodo, Please find the complete protocol as an attachment
Your attachment was helpful to our research, Dear Sanjeev Kumar. Thanks.
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