Endogenous control gene for real-time PCR

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Sergi Castellvi-Bel
Sergi Castellvi-Bel's picture
Endogenous control gene for real-time PCR

Hi!
We are working with real-time PCR for a gene named PRL-3 apparently involved in metastasis of colorectal cancer. We are working with RNA from peripheral blood lymphocytes. Does anybody know a good endogenous control gene to work with? So far, we have tried beta-actin and TBP.
Thanks and best regards from Barcelona

smartee
smartee's picture
You can add synthetic

You can add synthetic kanamycin control RNA from Promega, cat#C1381, to your RT reactions, and then run different housekeeping genes to see which ones don't change. As long as you start all of your RT reactions with the same amount of RNA it should work.

badcell
badcell's picture
Hi. You can check this link:

Hi. You can check this link: housekeeping genes list
I always use beta-actin and tbp as housekeeping genes, though. What happened? Didn't they work for you?
Best regards from Barcelona, too!

nin1318
nin1318's picture
i am having a problem too

i am having a problem too with our real-time....i had tried 18S but that didn't work and i know that GAPDH doesn't work either...i am hoping to find one that someone reports on that i can be confident of the levels remaining constant. b-actin isn't likely to work because i'm looking at neurons and expecting synaptogenesis so actin is likely to go up....

badcell thanks for the housekeeping genes list.

smartee
smartee's picture
18S RNA is a pretty lousy

18S RNA is a pretty lousy housekeeping "gene." Contrary to the popular opinion, there usually are irregularities in the amounts present in different samples. It is best to use one or two exogenous controls, and to test various housekeeping genes against these to sort out those that don't work under your specific conditions.

adagnall
adagnall's picture
we use GAPDH, and it seems to

we use GAPDH, and it seems to work well.

sentinel
sentinel's picture
I think may be you can run a

I think may be you can run a PCR and then agarose gel first to see any signal before taking it to real-time PCR.
Do you use SYBR green or TaqMan? TaqMan is accompanied with exogenous control.

ToNoFo
ToNoFo's picture
What do you mean you have

What do you mean you have "tried" control genes. Do they not amplify? If so then the problem is in your sample preparation, as most control genes are expressed at fairly high levels (say Ct's of 12-16 on an ABI). If you are getting something different, then your sample is your problem.

Sergi Castellvi-Bel
Sergi Castellvi-Bel's picture
ToNoFo wrote:What do you mean

ToNoFo wrote:

What do you mean you have "tried" control genes. Do they not amplify? If so then the problem is in your sample preparation, as most control genes are expressed at fairly high levels (say Ct's of 12-16 on an ABI). If you are getting something different, then your sample is your problem.

Thanks all for your comments. By saying that I have tried some endogenous control genes I mean that after using them I don't see a similar behaviour in terms of amplification efficiency. Buy, hey, news update...I am using GUSB now and it works wonders for our samples and target gene.

Happy holidays!

nin1318
nin1318's picture
ToNoFo wrote:What do you mean

ToNoFo wrote:

What do you mean you have "tried" control genes. Do they not amplify? If so then the problem is in your sample preparation, as most control genes are expressed at fairly high levels (say Ct's of 12-16 on an ABI). If you are getting something different, then your sample is your problem.

i tried 18S it and got significant difference in the levels between my control and experimental samples. that wasn't necessarily surpising though since i'm inducing huge increases in transcription and translation.

it seems as though every housekeeping gene that has ever been used has been seen by others to have varying levels depending on the experimental paradigm. i have recently been turned on to one for use in rodent visual cortex which hopefully will work. so far the preliminary results look good, but i have only had a chance to run it once. it's called HPRT, or hypoxanthine phosphoribosyltransferase.