Minimum amount of RNA for real-time PCR - (Mar/10/2006 )
What is the minimal amount of RNA to use for a TaqMan real time PCR reaction? Because I have just a very low amount of RNA isolated from my samples (just 1 microgram).
I have similar issues to you, so I may not be able to give you the best advice.
What I have noticed is that it depends on a lot of variables. Like, the copy number of the gene of interest in your sample, the quality of the RNA, and whether its from a plasmid or just genomic RNA that you have extracted.
Someone else might be able to help more specifically. Can you tell us something about those variables?
I've made cDNA using just a couple hundred nanograms of RNA in 20uL reactions. A single uL of this first strand reaction was sufficient for qRT-PCR of the genes I needed to look at. Generally I would use 1ug RNA per cDNA reaction but haven't seen any problems using several fold less RNA if it becomes necessary.
If you are still concerned look into the protocol designed for single cell RT-PCR, from Ambion I believe. They perform an amplification step to increase starting material.
Thanks for your answers so far. To add a bit more information about the experiment: I isolated RNA from FACS sorted cells and obtained 0,6 microgram total RNA, which I have to use after RT for a 364 well TaqMan real time PCR. The quality is quite ok, but a have to measure low abundant genes. So any further hints would be nice. Thanks.
I think you might be really struggling to make enough cDNA for 364 wells from just 0.6 ug RNA.
Even if you did very small reactions, and added only 0.5 ul of cDNA to each... you would still need 182 ul of cDNA, which for me would normally mean around 9 ug of RNA.
Can you do a practise run and see what is the minimum cDNA you can use?
Have you thought about amplifying your cDNA in some way?