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Dear all, please help me! I really really need your help!I intended to compare the expression level of some genes at different time point in my target organism, I choose to use 18s rRNA gene as the internal standard for a rough determination of the target gene expression level.
I had adjusted the total RNA I used for RT-PCR to the same quantity based on OD260 of the total RNA samples, and the bands of the PCR product of 18s rRNA looked almost the same lightness on the electrophoresis gel, however, the expression level of the target genes appeared to be in great fluctruation which was beyond our expectation.
A senior researcher in my lab told me that 18S rRNA was not reliable for there were too many copies of them in the total RNA, and the only thing I could relied on, if I have to use ordinary RT-PCR other than real time PCR, is the quantity of total RNA I used for Reverse Transcription reaction.
I am really puzzled! If 18s rRNA is not reliable, why some people choose it?! What should I do then?
Can't I use image analysis software like ImageJ to caculate the ratio of my target gene to 18s rRNA and compare the ratios at different time point so as to decide the change in gene expression level??
Edited by biobunny, 28 August 2009 - 05:35 AM.
