RNA concentration is too low to detect gene of interest by qRT-PCR - (Jun/15/2014 )
I've got a basic question in qRT-PCR. I am new to that but I am sure that many of you faced this problem before.
I am just simply need to do a qPCR but my samples' RNA concentration is very low (ranging from 2.7-1.6ng/uL).
As I calculated, the maximum amount of cDNA in a 10uL qPCR reaction would be 0.549ng.
Before measuring the genes of interest, I first run the housekeeping gene Gapdh to see what is the Ct value in order to decide should I go ahead for other genes. I've got 23-28 Ct among the samples. So, it's already close to 30.
I am sure that the expression level of my genes of interest are much lower than Gapdh (10-15 Ct behind).
Now, I think that I could not get reliable or even cannot get a Ct value of those genes.
So, is there any method that I could detect those genes?
Isn't it concentrating the RNA is the only way?
You have 2 alternative:
1. place samples in a speed vac and concentrate them
2. re extract them.
What type of sample you are using and how you extract the RNA? It appear too low for the majority of protocols.
And too low to be even measured accurately.
I think the RNA level you recovered is still pretty high and I suggest you can review some protocols for single cell RT-qPCR. Meanwhile, of course I don't think every gene expression can be measured at very low level of RNA. We have worked on single cell qRT-PCR for a while and published some manuscripts including the following:
Gao et al. 2011. qRT-PCR based quantitative analysis of gene expression in single bacterial cells. J. Microbiol methods. 85:221-227.
Shi et al. 2013. Monitoring single cell stress response of diatom Thalassiosira pseudonana RT-qPCR. Applied Environ Microbiol. 79(6):1850-1858.
I had the same experience as in the past months I recovered more or less the same concentration of RNA from my samples.
I fixed the problem in two ways:
First I bought a kit from Qiagen specific to recover RNA from "difficult" samples and/or from a very low amounts of cells. The kit is the RNeasy Plus Micro Kit.
Second if you have still some samples from which you have extracted a very low concentration of RNA and you need to analyse with qPCR, you can run a pre-amplification step on your cDNA before doing the real qPCR.
I don't know precisely how is the cycle to have to set on the thermocycler to do Pre-amplification, because I didn't the PCR personally, but i am sure if you search on internet you can find something.
Hope this can help you.