In my experiment i treated my sample with a compound. i want to see its gene expression of 2 target genes. I used 16s rRNA gene as reference. the average Ct value of untreated is 8 while the average Ct value of treated sample is 14. Ia the difference of Ct value is fine? How can we normalize our target gene with the reference genes?
Are you sure that your reference gene is really a reference gene? I recommend you the following paper:
http://download.springer.com/static/pdf/393/art%253A10.1007%252Fs13353-013-0173-x.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs13353-013-0173-x&token2=exp=1441685184~acl=%2Fstatic%2Fpdf%2F393%2Fart%25253A10.1007%25252Fs13353-013-0173-x.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs13353-013-0173-x*~hmac=43c2eb97252ec485a5ed858da3d419d248c7ba2bdb49c8a1089af6731992bd92
In addition, please check the following paper:
http://qpcrupdate.com/bustin-2000.pdf
Hope you you find this useful
Dear Ahmed,
Try to use the know conc of 16s rRNA gene, you may clone any portion in to a cloning vector and run each time as control.
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