semi quantitative PCR - quantification of gene expression (Apr/18/2005 )
i need some explanations on semi quantitative pcr. I tried to analyse gene expression without quantitative pcr (my lab is not equipped) so i stop pcr at some cycles (e.g. 25, 27, 29, 30, 35th cycle) and i analyse pcr products on an agarose gel. But i found that between 25 and 35 dna longer reduced from 850 to 650 bases. I don't understand. Have you ever got such results ?? Do you have some "easy way" to advice me ?
Thanks in advance and hope someone will be able to help me.
Could it be that the smaller amplicon is a non-specific product that reaches detectable levels only after several amplification cycles?! If not, you might want to check the final concentrations of the ingredients used in the PCR, (But, I sincerely doubt that as the cause for your observation!)