Taqman assay for detecting expression of genes carrying specific SNPs
Posted 26 July 2012 - 10:34 AM
I have some genes present in multiple copies and I want to detect expression of only the ones having specific SNPs.
I was told that Taqman can do the trick so I looked about it and found that there is a number of different Taqman kits.
I'm thinking that I should deal with this by first getting cDNA from my RNA and then using either the Taqman SNP genotyping or Taqman mutation detection kits to quantify my mutant transcripts.
Am I right? What different does the Taqman Expression kit has to offer? What's the difference between the SNP genotyping and mutation detection kit? Is it just that the latter can be used to also detect mutations other than SNPs (e.g. long indels)? Since my gene is not in the list of predesigned experiments, can I give my genes to a vendor and have them design the primers/probes? Can I design them myself? If so, is there a guide about the features my primers/probes should have?
Posted 26 July 2012 - 09:42 PM
Usually companies that synthesize primers can help you with such designs. They will charge you for it, but not much. It's their job after all and they know what to do. However, if you are a student it would be best to do it yourself. There are some guides on protocol-online.org. It'll take you some time to read and learn. you need to take care about the amplicon size, primer Tm and stuff.
If you could tell me which company you are buying your kit from I can read the manual and help you more. Most of these kits work according to melting temp. The PCR machine software allows you to compare melting temp of your products to find which one of your samples is mutated.
Posted 28 July 2012 - 12:48 PM
Mutation detection kits are designed to detect even a very minor variant, where increased sensitivity is needed, so these are castPCR assays, with allele specific primer and MGB blocker for the major allele.
However none of these kits are intended for cDNA.
As I understand you want to know if your minor SNP-containing transcript is present, so the mutation detection approach would be better, but you probably don't need castPCR because your transcript in not that rare.
I would go for allele-specific PCR (specific primers on the SNP) with LNA spiked nucleotides at the 3' end, that increase specifity of the amplification. I don't know of any free tool that allows to design this, because this is kind of tricky when calculating the right position and Tm. So company design would be better. All companies selling LNA oligos bought patent from Exiqon, so Exiqon is probably the first one to ask for price. Some companies can design for free if you are buying more expensive oligos, for example Roche does that.
I never trust anything that can't be doubted.