2 replies to this topic

[Spl33n]

Hello all!
I am an undergraduate medical student working on a project in our molecular genetics lab (please excuse my lack of knowledge in molecular biology techniques!).
We have RNA samples from three individuals, and I would like to verify whether disturbance of normal splicing has taken place in these individuals. I have designed three pairs of primers aligned on mRNA transcript sequences of our gene of interest, and all I want do is to examine whether alteration of RNA transcript length has taken place (due to loss of exons, or retention of introns in the transcripts).
I have noticed that there are three ways to study mRNA (hexamers, oligo-dT, gene-specific RT-PCR). My question would be:
Is it possible to perform an RT-PCR reaction with ordinary primers in a similar manner to PCR (is this the gene-specific RNA)? Is it absolutely necessary to construct a cDNA library to perform my investigations?

Thank you for any info in advance!

[bob1]

1) yes.

2) yes.  Think about how PCR works.

[Spl33n]

darn:) You are right
You have to use RNA dependent DNA polymerase (RT) to convert RNA -> DNA
And then you can perform cycles with DNA dependent DNA poylmerase to do DNA-> DNA amplification
I thought I can force RT polymerase to do all the work for me, but I can't just make it have DNA dependent DNA polymerase activity:)
Silly me.
thanks!