Real Time vs Traditional PCR results - (Feb/25/2010 )

Hi,

I am new to this Real Time PCR. I am doing a two step SYBR green assay. Both my house keeping gene (18s ribosomal) and my experimental genes amplified well, the CT values for my housekeeping gene were all within the acceptable range, and the dissociation curve showed only one peak for both genes. So my question is when I performed the Real Time assay and a traditional PCR side by side, they gave different results. Has this happened to anybody else? Any suggestions?

Thanks!

Hi Fed Up with PCR,

It is not uncommon for real time PCR and standard PCR to give different results. Unless you are doing exactly the same thing with both techniques, then you will likely get different results.

I assume that by different results you mean to say that with one technique you get higher expression of your gene, and with the other technique you get lower expression. In order to understand what you are seeing, it would be useful to know what are the Ct values of your 18S and GOI assays, as well as to look at how you used your 18S assay in standard PCR to normalize the bands you saw with your GOI assay.

Ultimately, how many replicated did you run? If you have at least 3 biological replicates for each experiment and you still get different results with both techniques, then that is hard to explain (assuming you are using the same cDNAs for both standard PCR and real time PCR). My guess in that case is that your standard PCR assays are not really detecting the same things as your real time PCR assays.