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Large Difference between Ct of 18s rRNA and Target Gene - (Feb/17/2010 )

Hello,

I have been looking for decrease the cycles between my target and 18s rRNA, but have not had much luck. I was hoping that some of you would have experiences with this.

I have been using geNorm to select the housekeeping genes that work best in our experimental conditions. 18s and beta actin seem to be the most consistent and stable across our treatment.

Right now, we use 40 ng of RNA to make our cDNA, and then we dilute the cDNA 1:50 for our qPCR reaction. Most of the target genes and beta actin have Ct values from 25-30. 18s has a Ct value around 16. Can I use the delta delta Ct method with these conditions? Or does the 18s rRNA value have to be closer to 25?

If it is not good practice when one compares the gene of interest to the housekeeping gene when there is this much of a difference, my question is why?

Also, does anyone have any suggestions on how to get the Ct values closer together? I know I could dilute the cDNA that is going into the wells with the 18s rRNA further, but because of pipetting errors and other variables, I would rather not go this route. Any suggestions?

Thanks for your help.

-JonBio-

House keeping genes, i have seen in my expts always have an increased expression when compared to the targets.Hence it is obvious that you see Ct differences between target and endo. And for expression studies, the targets are normalized with the HK to eliminate any error between the treatments/samples.
For checking pipetting errors, is there any passive reference included in your reaction mix? i use ABI 7500 system and the sybergreen what i use has a passsive ref included in my reactions.
Ct values for bettter results should be between 15 to 25 cycles. Anything beyond this is doubtful.
I do not agree with dilution of samples only in HK, because it will effect the overall expression pattern, if you are using ddCt algorithm.

Hope this helps

-Primrose-

JonBio on Feb 17 2010, 04:25 PM said:

Hello,

I have been looking for decrease the cycles between my target and 18s rRNA, but have not had much luck. I was hoping that some of you would have experiences with this.

I have been using geNorm to select the housekeeping genes that work best in our experimental conditions. 18s and beta actin seem to be the most consistent and stable across our treatment.


Best practice for QRTPCR is to pick housekeeping genes that have a similar expression level to your gene of interest (I'd say no more than 5 cycles different would be ideal). Look through some more expression databases and find a housekeeping gene that is expressed at a lower level. In particular rRNA is always a poor control to use if your gene of interest is an mRNA (RNA preps often enrich either for rRNA or mRNA unpredictably so it's hard to compare across samples).

It's totally not acceptable to dilute the sample that goes into the HK reaction. You should load exactly the same template into both your reactions.

-jojoziggy-