spiking cDNA or PCR product for standanrd curve dilutions - (Aug/26/2008 )
One of my HSP RNA seem to be very low in my control samples. All this time I have used these control samples to generate standard curve dilutions, but in this case, with lower dilutions I dont get the expected values or curves dont come up. Howver control samples at a different time period shows upregulation.
My questions are
1) is it OK to use these control samples at a different time period ( affected by time.etc) to generate standard curve
2) is it Ok directly use PCR product of a higher dilution or spike my control cDNA with PCR product to generate standard curve
both of your options should be ok.
maybe the second one is more feasible because you can make your standard curve over a broad dilution range (very important!) but the "ambience" is comparable to your unknowns. I'm doing artificial standard curves over a dilution range of 6-7 logs with triplicates. I think with diluted cDNA this could be only achieved with 18S....
but the most important rule with standard curves is that your samples must lie within your STD curve range i.e. extrapolation is not acceptable.