Is this contamination? - (Mar/22/2007 )
My student set up standard curves for a gene of interest and a house keeping gene.
For the gene of interest, she is getting a slight shift in Tm between the highest concentration (83.6deg) and the subsequent dilutions (82.4deg). She is also detecting a product with a Tm of 82.4deg in her no-RT control. I have previously carried out qPCR for this gene numerous times before and it usually has a Tm of 83.6deg.
My student also ran the PCR products on a gel and is seeing a product of the expected size in both the serial dilutions and no-RT control.
For the housekeeping gene, all dilutions are melting at the same temperature and the no-RT sample is clean. She also ran a water control and this is clean for both primer sets.
Is this likely to be due to contamination of something? And if so ...... what??
SYBR green melting curve are know to fluctuate. so is not surprising your students is getting 82.4 and 83.6.
If you are curious about the PCR product, you may sent it for sequencing..... then you will know what courses the difference in Tm.
regarding to the band present in both serial dilution and non-RT control....
there are two possibilities:
1. your student contaminated RNA stock with DNA during the process of setting up PCR. or
2. contaminated RNA stock with DNA during extraction, especially true if she is using TRIzol to extract RNA.