I was wondering has anybody experienced problems with reproducibility when trying to get an estimate of gene expression using semi quantitative pcr. My situation is as follows. Im doing two step pcr whereby i Isolate the rna from cells and then measure this rna concentration on a spectrophotmeter. I then put in a certain volume of rna into a dna elmination and reverse transcripton step using the qiagen quantitect kit. I then use aliquots of this cdna for my pcr reaction. I am trying to examine expression of a gene with possible splice variants over certain time points so am using various combinations of primers for different exons of a published sequence of the gene. When i use the same aliquot of cdna and the same pcr kit and what i think i have done everything the same i get different band entensities when i then run the pcr products on a gel in order to attempt to semi quantify expression over my time course. I am hoping that somebody may have experienced a similar situation and found the solution. Interestingly when I use a taq man probe for another gene im interested in the reproducibilty between pcr runs is excellent so im not inclined to believe that its my pipetting that is the cause of the afore mentioned variablty. Any suggestions comments most welcome as im stumped and frustrated by lack of reproducibility!
problems with reproduciblity in semi quantitative pcr
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