RT-PCR amplification of an intron - has anyone tried this? (Dec/11/2005 )
Hi,
I was wondering if anyone has tried, or read, of RT PCR on a sequence within an intron?
I was sent a very sparse protocol on this method, and it's left more questions than it was supposed to answer.
Basically, i wants to amplify a small section of an intron when it's still RNA.
So, we know that'll be in low abundance, but i was wondering if there are *really good* methods of ridding my samples from DNA contamination.
I was planning on having a control of my sample not treated with with RT enzyme, to show that the amplification is from RNA only, not DNA. Any other ideas for controls?
Should the DNase (or whatever) be used before first strand synthesis, or after? In my normal real time PCR, it's with the kit, but my thinking at the moment is "because this is going to need more sensitivity, it'll need as much DNA removed as possible."
Anything else anyone can think of?
thanks a bunch.
vetticus
what you should do is separate the cytoplasm from the nuclei and only isolate RNA from the nuclei, eg prior to splicing
DNAse isolation should be carried out as part of the RNA extraction, so that the digested DNA can be removed from the prep
I have done this on the ETS and ITS regions of the rRNA precursor with good success
You use the DNase before reverse transcriptase, right after RNA extraction. As long as you save some of the DNase treated total RNA (i.e. don't use it all in reverse transcriptase) you should have enough.
I would recommend Ambion's Turbo DNase/Turbo DNA-free. It is noticeably better than the other products out there if you use it in high concetrations (I have used Amersham/Qiagen stuff as well).
There is no way I'd ever try to amplify introns (seems pretty futile), but good luck,
-Matt
As DNase doesn't completely digest the DNA, rather breaks it down into shorter fragments, for an application as unusual as this I would definitely recommend a purification step using a silica column type kit following the DNase digest in order to remove the digest products. Silica kits do not isolate anything less than 200nt, meaning partially digested DNA is removed
I was planning on having a control of my sample not treated with with RT enzyme, to show that the amplification is from RNA only, not DNA. Any other ideas for controls?
// I think u should digest all your RNA samples with DNase and purified again first. Then only u carry on with RT-PCR including a negative control (sample without RT enzyme). Correct me if i wrong.