I have been doing qRT-PCR for TNF gene and I saw that there was a difference in Ct values in the untreated cells over subsequent three passages. When I look at the reference gene beta tubulin it looks stable. What can be the reason for this change in Ct just over passages in untreated cells?
It also is important to take the samples at the same time of the day, because it is possible that the expression cycles over the day. Alot of genes are diurnally regulated.
With what kind of cells are you working? The expression of many factors varies from passage to passage because either when we are trying to simulate accurately the physiological environment, it is not the same as in the body; also when we trypsinize to subculture we make a selection of the most clonogenic cells, so the ratio of factors vary at every stage
It also is important to take the samples at the same time of the day, because it is possible that the expression cycles over the day. Alot of genes are diurnally regulated.
To me it appears the basic steps are not standardized and what I mean is , Have you done the efficiency test runs for untreated and the treated cells? All the factors have to be exact same in terms of concentrations? volume? run conditions? RNA prep? cDNA preps? These things are major points even though it is pain, in the end the results are gold.
Yes I do need to still do the efficiency curves, but I use pre designed primers from Integrated DNA technologies, and they seem to have a high efficiency. The quality of RNA seems fine, although I occasionally get solvent contamination and I only use these samples if 260/280 is more than 1.8. We use kits for RNA prep and cDNA preps which have been working preety consistent over the last 2 years and I never had this problem before. I dont see the same when I am culturing cells in the same passage and using biological replicates form the same passage , this happens when I use the subsequent passages for the same experiment?
I will highly appretiate if some one can send me a standatd SOP used in their labs for q RT PCR along with the EXCEL sheet you may be using to make your calculations. This will help me a lot. My email is [email protected]
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