Question

You decide to use RT-PCR to confirm your predictions about the exon organization of the 2 mRNAs in tissue such as heart and epidermis. Describe in general the experiment you would do, specifying where your PCR primers would be designed to bind. What results would you expect. Please be specific

Answer 1

RT PCR is also referred to as reverse transcription polymerase PCR. This is the most sensitive form of PCR, in which there would be detection of mRNA, and there would be quantification of this detected rna. The samples used in this technique are much more minute as compared to the othe techniques. There are two protocols for this method, one is one step and the second is 2 step process. In the one step RT PCR, there would be mRNA targets which have a size up to 6kb, which would be undergoing a process of reverse transcription, followed by amplification.

In RT pcr, the primers that would be used, will be affecting, both the specificity of the product and the size of the cDNA that’s being produced. there are different primers that would be used in RT pcr, which are:

• Oligo (dT)N which would be hybridising to a poly A tail, present endogenously. If there is a template that would be containing an oligo A stretch, the primer might be binding to this, which might cause misprintint.
• If an anchored oligo (dT)N is used, it would be generating a full length product of cDNA, and would be hybridising to the initial part of the polyA tail. This is the most preferred primer.

Random primers used are to be used at concentrations greater than 60 µM s that best results would be obtained. For amplification, there have to be presence of two primers of PCR, which would be specific for the gene of interest. Primer that are designed have to be such that there should be differentiation in the product that is amplified and the cDNA. The primers designed have to either be designed such that they would be annealing on Exons or on both sides of introns. As a result, the amplified product from the DNA would be greater than the product obtained from the cDNA which is intron less.

Alternatively, primers could also be made such that they would be present on the exonto exon boundary, and would be present across more than one intron.

Following this, there would be a choice of the enzyme, reverse transcriptase. In one step process,

• There would be denaturation of the template at 94degC following an increase with every cycle.

In the two step process, the denaturation occurs at 65degC for 10 minutes followed by use of primers and reverse transcriptase for synthesis of the first strand.