Question

Explain the basics of one step RT-PCR . Design an appropriate RT-PCR experiment to detect a gene’s level of expression.

Answer 1

In a one step RT-PCR, the conversion of RNA to cDNA and the amplification of the DNA is done in a single reaction tube. The reaction buffer is same for both the conversion and the RT enzyme used for the conversion of RNA to cDNA is carried out at a hoger temperature and the amplification of DNA is done at lower temperatures. The prinses used are gene specific primers.

The experiment setup of one step RTPCR is carried out in the following way:

1. Fresh RNA needs to be isolated

2. Then the components of the one step RTPCR reaction mixture should be mixed in one tube as per indication on the kit.

3. The reaction needs to be set as per the primers anne aling temperatures. The annealing temperature for primers during RNA to cDNA should be set at higher temperatures and the amplification of the DNA done at lower temperatures. Hence the amplification of the DNA tells us the level of gene expression inside the cells.

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