RT-PCR for an intronless gene - (Mar/02/2005 )
I want to know how to perform a RT-PCR for an intronless gene. I've never done it before. I need helps!
nobody can help me?
I would say, make sure you treat your RNA with RNAse-free DNAse and then when you go an perform your PCR, make sure you run a no-RT sample to ensure amplification is not from gDNA
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.
the difference is that you can't use intron/exon boundaries in the design for primer and probe(s). so your system will be able to detect residual gDNA as well as reversely transcripted cDNA. therfore you have to include the DNase step methylnick and fred_33 suggested. In my TaqMan system this reduced the gDNA copy number from 3E5 to roundabout 50....(remember that enzymatic reaction are never 100% quantitive)
i am not very sure but may be this could work for you. Your mRNA supposedly have poly AAA. so desigin one primer with inclusion of poly A. because genomic DNA will not have these poly As. Another way is check for the adjusant gene or intergenic region. so forward primer should be with in the gene and reverse primer crosses intergenic region it should be somewhere 5'UTR of other gene. this way you can see the difference between genomic and cDNA. good luck.
my two cents..
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.