3 replies to this topic

[Dan Multer]

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?

[cashlern]

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)

[Dan Multer]

cashlern, on 10 August 2011 - 12:04 PM, 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?

[Trof]

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.