mrna detection with sequence specific primers - (Oct/08/2008 )
Dear all,
I want to start working with RNA and I want to detect a certain mRNA in mouse brain tissue.
Just to see if the mRNA is present in 1 or 2 splice forms (with or without exons 2 and 3)
the mrna is at the most 441 AA (MAPT) long and I want to amplify a sequence in the 5' region (exon2-3), actually primers are designed in exon 1 and 7 to yield a amplicon of about 600 bp with both exons and 400 without those 2 exons. (normal RT-PCR, not quantitative)
I see that everyone uses random hexamers or oligo dT for conversion of mRNA to cDNA.
But i find little or no information on doing that with seq spec primers. Seq of the primers is now about 57°C, but by shortening the primers i could get a lower Tm.
Is it wise to start directly with seq spec primers (gives more specificity + higher yield in my opinion) and howand with wich RT enzyme do i do this. most enymes work around 25-35 or 60°C.
Or is it better to start with random hexamers or oligo dT primers and work with seq spec primers on the cDNA?
Does it matter how I isolate my RNA, isolate total RNA or search for mRNA isolation kits?
thank you very much
I'm not an RNA guy, but your sequence-specific primer approach sounds OK to me. I would design primers that cross an exon-exon boundry so as not to amplify contaminating genomic DNA, and recall that the Tm of DNA-RNA hybrids is different from the Tm of DNA-DNA hybrids (see the reference note here).
Thx, at least I know the approach might work, now some more details.
Can anyone tell me if the cDNA production with seq, specific primers is diferent than with oligo dT or random hexamers (Tm, enzyme,...)
Do I adjust my primers Tm to the working temp of my reverse transcriptase or can I use normal size primers (afraid that if primers are to short I will pick up multiple annealing sites)
The primers are in exons and the introns are so large that if I would amplify gDNA with these primers I end up with an >170kb amplicon, wich I doubt will happen, (So I am safe there
)Does anyone knows good kits from either Westburg, Qiagen, promega or so that I might need?