# internal standard - (Jun/07/2010 )

I am an absolute beginner in the Tag-Man Real-Time quantification of gene expression. Can anyone explain to me how to check if a reference gene that I chose was appropriate in my experiment? I did test of efficiency of target and reference genes across different dilution. How I can to verify that the efficiency is close and if I can use the multiplex reaction?

-ksenija-

A good rtPCR machine should calculate efficiency from your dillution series (standard curve). Or you can use the formula
E = 10<–1/slope> and get slope from plotting the Cts and concentration in the Excel graph.

To know if you chose the right housekeeping gene there should be minimal differences in quantity when using the same amount of cDNA. There is no easy way to tell if it's good or not, but you can choose several housekeeping genes and use software like geNorm or BestKeeper to determine the best of them.

As far as I remember, in making the multiplex the same efficiency is not that important (but of course it must be within the "good" range 90 - 110 %), but Ct's for each of the genes should be similar. If they're not, use more primer for the higher Ct gene (or less for the lower Ct gene), so that the quantities of each gene is approximately equal.

Use this site, to get more resources about quantitative PCR.

-Trof-

Trof on Jun 7 2010, 03:04 PM said:

A good rtPCR machine should calculate efficiency from your dillution series (standard curve). Or you can use the formula
E = 10<–1/slope> and get slope from plotting the Cts and concentration in the Excel graph.

To know if you chose the right housekeeping gene there should be minimal differences in quantity when using the same amount of cDNA. There is no easy way to tell if it's good or not, but you can choose several housekeeping genes and use software like geNorm or BestKeeper to determine the best of them.

As far as I remember, in making the multiplex the same efficiency is not that important (but of course it must be within the "good" range 90 - 110 %), but Ct's for each of the genes should be similar. If they're not, use more primer for the higher Ct gene (or less for the lower Ct gene), so that the quantities of each gene is approximately equal.

Use this site, to get more resources about quantitative PCR.

Is it required to check a few housekeeping genes. May I, on the basis of literature to choose one? How in that case I can know if it suits to my experimental conditions?

-ksenija-

It is possible to choose one from literature or common used one, but unless they validated it in similar experimental conditions (same treatment, doesn't need to be same genes of interest) there's no way to tell if it's a good housekeeping gene for your assay. Theoretically if you get very similar Cts for all the treated samples while using the same amount of cDNA, then your housekeeping gene isn't affected by treatment and can be used. But if you're not sure, or have a different treatment, then it's better to choose several (published) stable houskeepers and run a stability experiment.

But I think most people don't do that, they just take GAPDH or beta-actin and use it right away.

-Trof-

Trof on Jun 10 2010, 02:16 PM said:

It is possible to choose one from literature or common used one, but unless they validated it in similar experimental conditions (same treatment, doesn't need to be same genes of interest) there's no way to tell if it's a good housekeeping gene for your assay. Theoretically if you get very similar Cts for all the treated samples while using the same amount of cDNA, then your housekeeping gene isn't affected by treatment and can be used. But if you're not sure, or have a different treatment, then it's better to choose several (published) stable houskeepers and run a stability experiment.

But I think most people don't do that, they just take GAPDH or beta-actin and use it right away.

For the pilot experiment I chose one housekiping gene and several candidate genes for the determination of their expression. Some candidate genes (in control samples) have similar Ct value as a reference gene while the other varies from 5-10 cycles. Even hausekiping gene has a higher Ct value of the same candidate genes. Is that right?

-ksenija-

Using housekeeping genes from commercial panels right away without validation is non-sense. The general consensus is to validate reference genes for the conditions in which one is working.
There's a new tool out there called RefGenes that helps you to find genes that are most stable in a particular tissue type. It processes data from thousands of microarrays and is very fast. RefGenes is open access and freely available at www.refgenes.org.

-phz@nebion.com-