I am performing relative quantification of NOS2 gene in PBMCs isolated from human blood samples using SYBR green detection method. Ct values are in the range of 38 and 40 with very good amplification and melt curves. The gel picture of amplified product is specific to the target gene. Are these Ct values suitable for analysis of gene expression studies? The expression of target gene is downregulated in disease group in comparison to the control group. On the other hand, the end product of this gene is elevated in disease group than control. How can I support these findings?
The critical point here are the controlls, you need a good controll to proof that your signals are not a result from DNA contaminations. Did you test that using a minus RT controll? (No reverse transcriptase in cDNA synthesis) Can you use more RNA for cDNA synthesis?
I have not used minus RT control. RNA concentration is 1 microgram and to eliminate DNA contamination, i have included Dnase treatment step in my protocol. I have been using the same cDNA for other genes and Ct values are less than 35 except in NOS2.
Well I would always recomment to do a minus RT control, you cannot be sure that all the DNA is gone.
What kind of cDNA dilution do you use for qRT-PCR?
Thanks for your suggestions Mikael and Ulrike. It is quite possible that there is no expression of NOS2 in PBMCs. Now it is clear to me that we should not rely on these Ct values.
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