I have patients and controls who share exactly the same heterozygous mutation in one of their protein. Controls are obviously healthy- So my plan is to see which allele is prominent when it come to protein expression.
I'm wondering if I can design TaqMan probes for both mutation and for WT region and do real-time PCR to see if there is difference in the expression of mutated RNA between the controls and patients.
Please let me know your thoughts!
Thanks
probably you would be better off with Simple Probes from Roche (or TIB-Molbiol.de).
These are hybridization probes that emit light only when bound. At the end of the reaction you make a melting curve where you obtain two different peaks for the amplicon with and without the SNV. They work best oon the LightCycler since it makes better melting curves than other machines.
Simple probes are absolutely easy and reliable for SNV analysis
Yes, entirely possible.
I'd suggest to design two mutation specific probes (hydrolysis, like e.g. TaqMan) that will bind only to a specific allele und label them with different fluorophores (there are tools available that can help you with that). In that way you can multiplex them in the qPCR (which won't work with SYBR) and simultaneously measure the quantity of both alleles (by comparing the Cq values) in the exact same sample which will reduce time, costs and spending of precious samples. If multiplex does not work or proves to be too difficult to establish you can still switch to singleplex.
It will take a while and some effort to validate and optimize your probes, though, e.g. in terms of efficiency, cycling conditions etc.
And please don't forget to establish a solid, reliable and relevant normalization strategy BEFORE running your actual experiments. Also, consider the MIQE guidelines (see attachment) when planing and reporting your experiments.
Best regards
Yes, theoretically, and TaqMan probes are great. However, qPCR is tough to interpret and I've recently become a believer in digital PCR. Do you have access to a ddPCR? Might give you a cleaner answer. And, of course, I assume you've considered another gene(s) in the difference in penetrance. I've got a lot of unpublished work on a mystery I still haven't figured out. Humans are so complicated...
Hi Lori,
Couldn't agree more. But unfortunately No. All we got is a 5-plex PCR machine. This is indeed the first time I'm trying to use TaqMan probes to see the differences in penetrance.
The problem is the controls. Talk to the TaqMan folks - I had great luck with them. They will design everything for you, and hopefully you will feel confident in the results. While I had great probes, I never felt confident that the differences were real because the 'advertised' control genes, in my opinion and others, can't be counted on for consistent, stable mRNA levels. Try to talk to others who use the same tissue and see what they think.
Hi Lori,
I have always been perplexed by the use of normalisation gene. If I use probes targeting the WT and the SNP on my mRNA of interest, would I still need to use a normalisation gene (HK)? All I need is to compare the expression of the allele with the SNP to WT allele.
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