qRTPCR problem

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Maddy
Maddy's picture
qRTPCR problem

I am characterizing a cell  line (CHO) for mRNA expression of 4 different genes.

I performed qRTPCR to quantify the mRNA levels using 4 different primers sets and also b-actin (reference gene) primers.

But I do not have any control to compare/relate with it, and also i hav not isolated these genes, so cannot perform a standard curve for absolute quantifications.

Is it possible to use qRTPCR for knowing the relative levels of these genes? if so How can it be done?

Otherwise which other quick technique will help me out here. 

Biju
Biju's picture
Hi Madhavi

Hi Madhavi
You should be able get commercially available cDNA from standard CHO cell line. American Type Culture Collection is a good place to start with. Additionally , you could also get from a lab with well characterized CHO lines. Use of well characterized cell lines are important due to the possibility of contamination and also a requirement for publication in international Journals.
Isolation of genes is not needed for absolute quantification and the absolute levels are not always need to be determined for cell lines in existence for a while.
Biju Joseph

Maddy
Maddy's picture
Thanks Biju

Thanks Biju

How can I compare amongst those 4 different mRNA by qRTPCR? Does it not need any control? is it even possible?

Are you refering to the cDNA of well characterized CHO cell lines for control or for the standard curve?