I am planning to do a loss of heterozygosity (LOH) analysis for the CDH13 gene at different polymorphic microsatellite markers in ovarian tumor tissue. Can anyone suggest which is the better option for LOH analysis: conventional PCR or Real time PCR?
Real time pcr is a better option in the type of study u wish to perform, as u can compare levels and this excercise will yeild actual quantitative values if u also run standard after cloning ur gene of interest, and doing rt pcr for the standard, first.
Real-time PCR works:
Wilhelm J, Pingoud A, Hahn M. Detection of p53 allele deletions in
human cancer by quantification of genomic copy number. In:
Meuer S, Wittwer C, Nakagawara KI, eds. Rapid cycle real time
PCR: methods and applications. Berlin: Springer Verlag, 2001:
159–70.
Real Time PCR works better than conventional: Please see this article as follow
Application of a Modified Real-Time PCR Technique for Relative Gene Copy Number Quantification to the Determination of the Relationship between NKX3.1 Loss and MYC Gain in Prostate Cancer.
May be it is not relevant for your purpose, but LOH allows to know which chromosome or allele is loss, this could be interesting. But You need to keep in mind that with LOH analyses you identify a loss of heterozygotie ( for example allele A more represented than allele B) but you do not know if it's chromosome with allele B which is deleted or if it's chromosome with allele A which is amplified. Real time PCrRcould give this information.
Real time PCR is indeed theoretically the best choice. But here keep in mind that you wish to amplify polymorphic microsatellite markers. These will show different size then different intrinsic amplification efficiencies between the two chromosomes. Then you need not only a healthy tissue match control with a 1:1 copy number of each marker to see if there is LOH in tumor. I think you should also produce a set of mixes of alleles at different ratios (say 1:8 , 1:4 , 1:2 , 2:1 , 4:1 , 8:1 for instance) as a set of standards if you want to be quantitative.
PCR it self is a stochastic process, always comes with some intrinsic variation. If possible go for exact count method. If it is PCR, you have to design your primer very carefully ( Faulty design of primer some time end up with strand bias, that may appear to be a case of lOH. This may give you some idea, how those bias can happen. https://www.researchgate.net/publication/233538525_Differential_haplotype_amplification_leads_to_misgenotyping_of_heterozygote_as_homozygote_when_using_single_nucleotide_mismatch_primer.
Article Differential haplotype amplification leads to misgenotyping ...
@ Jerome Buard thank you for the suggestion. Please suggest how should I make the set of mixes of alleles at different ratios?
actually I prefer to use Real Time PCR , you can use FRET probes fro such application or HRM if you have RG6000 , so according to what you are looking for , is it Gene Expression assay or not but both could be done with very nice data on real time PCR
Real time PCR. Check a WT to normilize your data (without CNV or duplications or deletions)
I think appropriate markers labelled with radioisotope amplified by conventional PCR and separated by polyacrylamide gel will give good result. Visualization is important to see two separate alleles. Obviously u have to take matching normal.
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