real time standards

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Aaron Amano
Aaron Amano's picture
real time standards

I've been doing real time RT PCR and I've been wondering if it would be at all possible to apply one standard to all genes assayed...considering they all came from the same RT mix? Maybe applying the standard from the housekeeping gene? We have been using 18S as an internal control.

Tony Rook
Tony Rook's picture
Aaron:

Aaron:

Maybe the following site will help you out!

Robert M. Powell PhD and Tim Howel MRCP, Edited by Mary Span
An improved 18S ribosomal RNA control for normalisation of quantitative real-time PCR.

http://www.eurogentec.com/module/FileLib/RIBO-SOMALRNA-0503-V1.pdf

SanDiablo
SanDiablo's picture
When you say apply the same

When you say apply the same standard, are you talking about the standard curve or the internal control?

You may use one internal control to normalize between individual PCR reactions as well as to help you compare from one set of reaction (one run) to another....also between RT reactions. 18 s can be a good choice, but it is worthwhile to evaluate several "housekeeping" genes to see which best suits your system.

The standard curves must be run along side each corresponding gene you are assaying, (including your internal controls) otherwise you are not being quantitative. If you are just interested in relative quantitation, you can perform a delta delta Ct without running standards, (I do this occasionally when my standard curve fails) but having a standard curve tells you a lot about efficiency and the success of the reaction. Without a standard curve, you cannot be certain your gene of interest is present within the range of linear amplification.

Pharmalance
Pharmalance's picture
could you please provide more

could you please provide more details for your question (if its not confidential). what are you doing exactely (relative or absolute quantification, gene expression?) and what do you want.

saswati1
saswati1's picture
HI,

HI,

If you treat the cells with (say) some drugs and you see no changes in GAPDH or b-Actin expression level ( very common standards used for realtime), you can use either of them as a standard for all the genes.