Gene Expression Issues - (Aug/08/2011 )
Is it possible for a sequence to have a higher expression than 18s rRNA? I am looking at the expression of the Alu retrotransposon, and the cT values for some of the Alu sequences were earlier than the 18s. Is something wrong with my sample?
I don't understand your problem. Your question would suggest that 18s is the highest expressing gene in a cell? You certainly could have a target sequence with a higher cT value than an 18s target (assuming you load the same amount of mRNA for both)
cashlern on Wed Aug 10 20:04:55 2011 said:
I don't understand your problem. Your question would suggest that 18s is the highest expressing gene in a cell? You certainly could have a target sequence with a higher cT value than an 18s target (assuming you load the same amount of mRNA for both)
But doesn't rRNA make up >80% of the RNA in cells? Since 18s is a rRNA, I thought that it would have a very high expression. Could the Alu sequences have higher expression than the rRNA? Does the 293t cell line contain less rRNA than other cells?
You should know better than me the estimated number of retrotransposon RNA in your sample, but It wouldn't surprise me if they were actually more abundant. 80% is whole rRNA, so there's 28S and 5.8S too, it's not like there is 80% of 18S.
Other thing may be, if your Cts are really low (< 15), that 18S is so abundant it inhibits the reaction. If you dilute your sample like 500 - 1000x you may get Ct values in the safer range.