Use a internal control (Taqman probe) for qRT-PCR analysis like 18S or GAPDH (not preferred for cancer)

Hi,

Thanks for the answer. Can you explain a but what do you mean?

Hello Ella

If i have understood your question righlty, yes you can but you need a control cell line for the particular gene you are targeting.

If you are going to look at geneA in cell line 1,2,3,4 and 5. to say that they are upregulated or down regulate you need a control cell line with that geneA expressing are normal rate.

Control cell line gene is different from your internal control gene (hope you got that)

You can do absolute quantification also but you need to make (clone the gene to make std labour intensive) or get (from company expencive) your standards

Good luck.

Do you want to compare the expression of gene A to the expression of gene B?

Then the answer is: you will need to quantify both genes using an external standard (of known concentration). The reason for this is simply that same (d)Ct values for different primer/probe systems do NOT imply same concentrations. There are primer/probe-specific (and usually unknown) proportionality factors involved. These cancel out when comparing the expression of gene A in different conditions, but not when comparing gene A to gene B.

So you can NOT use dCt values. You must quantify all genes relative to appropriate  external standards (often called "absolute quantification"). If you use cell lines you likely can sufficiently well control the number of cells used for RT so you might not need normalization to different loads, but it is also possible to quantify a loading control (reference gene(s)) to normalize the results for the (effective) cell number.

Thanks!

That's exactly what I meant - to compare the expression of gene A to the expression of gene B.

I have 12 cell lines and I want to compare the expression of 7 different genes. In What external standard to you recommend to use?

Thank you,

Ella

You have to create that standard. The best practice is to clone the targets sequences, purify and linearize the plasmids, determine the concentration (e.g. by UV spectrometry), ditute it appropriately and use it as standard. Instead of cloning and using plasmids,you can make preparative PCRs and use the PCR product as standard. It is recommended to use "outer primers" for the prep PCR (not the very same primers as used for the qPCR).

Be careful and do not contaminate your qPCR lab with plasmids or PCR products. I recommend doing the standard preparation in a different lab (ideally in a different buidling!!) and take onyl the working dilution (with, say, not more than 1E6 copies/µl) of the final standard back to the lab where you set up the qPCRs.