RT-PCR for an intronless gene
Posted 02 March 2005 - 04:51 AM
I want to know how to perform a RT-PCR for an intronless gene. I've never done it before. I need helps!
Posted 04 March 2005 - 04:20 AM
All comments and communication are my own personal ones, and are not tied to any of my affiliations.
Posted 04 March 2005 - 05:20 AM
i don't think there's a difference between RT PCR for an intron less gene than a normal gene... Extract your polyA RNA, make the treatment suggered by methylnick, and normally there should be no problem.
Edited by fred_33, 07 March 2005 - 01:26 AM.
Posted 04 March 2005 - 05:59 AM
Posted 04 March 2005 - 07:55 PM
Posted 07 March 2005 - 08:03 AM
Any RNA prep will have gDNA/non-specific amplification; the issue is to keep it within reasonable limits. Intron-spanning primer design is not a cure-all.
When intron-spanning assays are designed, two issues are frequently lost: length of intron (if its within PCR rxn conditions to amplify, it may be amplified from gDNA) and pseudogenes (many genes will have processed pseudogene homologs inserted into the genome). The key is always to have no-RT control (along with other controls) included in your experiments.
no-RT controls should be at least N cycles away from 'real' measured reactions. I have seen people want to have N=10; you may decide on your own limit. Only then you can sleep tightly.