Can I use different amount of cDNA template to run real time PCR? - when the amount of templates are different,Can I use reference gene to (Aug/06/2007 )

Hi,all!
I have many samples to run real time PCR, but some samples have limited amount,so I use less RNA to run reverse transcription, then when run realtime PCR, I use the same volume of template .So my real time PCR have different template amount, some people said that Iｃａｎ　ｎｏｔ　ｒｕｎ　ｒｅａｌｔｉｍｅＰＣＲ　ｔｈａｔ　ｗａｙ，　ｂｕｔ　Ｉ　ｄｏｎ＇ｔ　ｕｎｄｅｒｓｔａｎｄ　，ｃａｎ　Ｉ　ｕｓｅ　ｒｅｆｆｅｒｅｎｃｅ　ｇｅｎｅ　ｔｏ　ｎｏｒｍａｌｉｚｅ　ｔｈｅ　ｒｅｓｕｌｔ？
ｔｈａｎｋｓ　ｆｏｒ　ｈｅｌｐ！
Ｊａｎｅ

Hi Jane,

I think it would be OK to run the reactions as long as you use a stable reference gene.

Using the same amount of cDNA is always better but even if you run reverse transcriptions starting with the same amount of RNA, transcription efficiencies may not always be the same. So in any case, you're not using the "same" amount of cDNA. That's why you're using the reference gene in fact, to compensate for any variations until the qPCR step.

So from that point of view, I don't see any reason why you shouldn't run the reactions that way.