I have been doing q RT PCR for almost a year now. I have seen that there are differences in reference gene ( Beta -actin and 18 s rRNA) Ct values in different passages (normal difference being ct values up to 4). This really is affecting my data. The cell line I am using is a bronchial epithelial cell line. We grow the cells to 100 % cofluency and begin the experiment a day after the cells are 100% confluent? Can any one tell me what may be the reason for this?
Did you try to optimised your HK genes with GeNorm? As this is first step before you start using Hk genes for qRT PCR. Try this with both genes along with other housekeeping genes and see weather these two genes comes up most stable genes.
But geNorm do not take into account for data coming from different treatments (passages in this case). There are more sophisticated free software to search for stable housekeeping genes such as NormFinder, BestKeeper, etc
We switched to L19 (gene encoding a ribosomal protein rather than rRNA) as a housekeeper and have had no problems with it across our human primary cells and cell lines. It's extremely stable and not affected by treatments/passaging. We tested with a battery of housekeeping genes and it came out the best for us. Also the advantage with using it compared to 18s is that it is also mRNA so we can use oligodT primers in our RT, plus the expression range is comparable to our genes of interest.
You can also try to put your candidate to reference genes in DataAssist (free software from Life Technologies), it is easier than Genorm and gives you a quickly graph of the most stable reference gene in your samples...Also, have you tried RPL13A as a candidate...you will have to test , but in my case it was more stable than 18s, GAPDH and beta-actin...good luck!
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