Human 18s rRNA - Human 18s rRNA and qRTPCR normalization (Feb/03/2010 )

Hi.

I am using Human 18s rRNA for normalization of my gene expression from human collon tissue from healthy patients as well as Chrone's disease patients. The procedure was pretty straigthforward. Extraction of RNA, cDNA synthesis and RT-PCR. The problem is that my Ct values for the 18s rRNA gene are very low (6-12). The manufacturer's (SABiosciences) insruction calls for a Ct value of about 18. The efficiency of the reaction is 94.5% and the R2 value is 99.9. Can someone explain this to me? Could it be that the gene is overexpressed. Coult it be contamination?

Thanks.

-dimitris31b-

Hello

How much of your cDNA are you adding per rxn?

Clare

dimitris31b on Feb 3 2010, 01:34 PM said:

-Clare-

5μl/20μl reaction

-dimitris31b-

dimitris31b on Feb 3 2010, 01:45 PM said:

5μl/20μl reaction

Try 1 ul or 5 ul of a 1/10 or 1/100 dilution of your cDNA. The template is probably just too concentrated. Doesn't mean it's overexpressed. Or better yet, run dynamic range (e.g., serially dilute 1/10 down to 10^-6, use each dilution as template) to figure out how much of your template you need to get a Ct of about 18.

-fishdoc-

I forgot to mention that I run a standard curve for the 18s rRNA and I had similar results. The serial dilutions were 5 fold until a final dilution of 1/3125. The desirable Ct value was reached at dilution 1/625. Is it practical to dilute the samples that much regarding the fact that the target genes show a Ct value over 20 without dilution.

-dimitris31b-

Hi dimitris31b

Perhaps this link would be helpful for your study. Please visit this link

-stylothecancer-

Hi again. Is it meaningful to dilute the reference gene differently than the target gene and still interpret the results correctly?

-dimitris31b-

dimitris31b on Feb 4 2010, 08:21 PM said:

Hi again. Is it meaningful to dilute the reference gene differently than the target gene and still interpret the results correctly?

Hi dimitris31b,

No.

-stylothecancer-