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RT-PCR for a one-exon gene?
Eva Amsen
Monday, 10 March 2008 21:23 UTC
I’m trying to see if I get knockdown for a gene for which there is NO available antibody to the protein, and for which I can’t design any RT-PCR primers because there is only one exon (so I can’t differentiate between genomic DNA or cDNA)
(Actually, even ignoring the RT-rules, there doesn’t seem to be any suitable primer pair within this exon either.)
Is there anything I can do (within a few weeks) to monitor knockdown, preferably at the mRNA level? (I am certain that the knockdown construct is present, as it’s fluorescently labeled and I’m FACS sorting, so I “just” need to know that it’s actually working) Obviously, raising an antibody to the protein would work, but sadly, I don’t have that kind of time, and it’s not that important, and I don’t get enough lysate to look at protein levels anyway.
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Replies
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Hello Eva!
I’m sure you’ll be able do design a suitable pair of primers for RT-PCR analysis. What you need to do to get rid of the genomic DNA problem is to tread your RNA sample, as soon as you get it, with DnaseI. This way you eliminate vestigial genomic DNA from your samples. Then, to control for that, you can use primers for another genomic region…some other gene to which you have primers on the introns for example….then you make 2 PCR reactions with a bit of your RNA (treated and untreated with DNaseI)…if you have a band with the untreated RNA and no band with the treated RNA your sample should be free of genomic DNA and you can proceed to the cDNA synthesis and RT-PCR analysis.
Good luck.
João
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Hi again,
Actually, in that procedure I suggested you can use the primers you design for RT-PCR analysis.
Take care.
João
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But how can I do the PCR to test it before I make the cDNA?
I’ll try the DnaseI suggestion: I’m isolating a large amount of control RNA from untreated cells this week, so I have some to experiment on. I might have some primers to mouse genes that don’t cross exon-intron boundaries (so they would work on genomic and cDNA), so I can test the difference between with/without Dnase.
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Hi Eva,
Welcome to our forum!
I’m showing my age a little here – but would an old-fashioned northern blot help? Although blots are less sensitive/quantitative than RT-PCR, some basic densitometric analysis of a carefully designed northern experiment might give you the info you’re after?
Anyway – best of luck with proving the knockdown, no matter how you go about it! And do let us know how you get on…..
Best,
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It needs to be quantitative. I’m looking at knockdown of a bunch of genes, and am already using RT-PCR for all the others.
I just came back to say that I suddenly realized the point of doing PCR before the cDNA (I was too tired yesterday, but now it all makes sense)
I’ll see if I have some primers lying around that can detect any mouse genomic DNA (I’m sure I do) and try those on samples with or without DNase to (hopefully) see genomic disappear entirely, and then I’ll order the primers to my tiny one-exon gene. -
Yes, that’s right. And even with those primers for your tiny gene the PCRs before cDNA synthesis must work. It’s very easy. Prepare DNA, tread part of it with DNaseI then make a PCR using treated and untreaded RNA and you should only have amplification in the untreated RNA.
Good luck with that.
João
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I tried this last week, and it seems to work! With DNase, there is almost no band from PCRing on the RNA sample, and without it I still get quite a lot from the genomic. Primers work too (I used a different primer design program and suddenly there were available primers)
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