Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

RNA concentration is too low to detect gene of interest by qRT-PCR

RNA concentration qPCR Low copy number picogram

  • Please log in to reply
4 replies to this topic

#1 kyliehm

kyliehm

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 15 June 2014 - 03:39 AM

Hi guys,

 

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?huh.png  



#2 merlav

merlav

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 56 posts
0
Neutral

Posted 16 June 2014 - 04:56 AM

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.


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#3 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,198 posts
109
Excellent

Posted 16 June 2014 - 07:25 AM

And too low to be even measured accurately.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#4 wxg118

wxg118

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 16 June 2014 - 09:14 AM

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.

 

 

 



#5 AleGasta

AleGasta

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 20 June 2014 - 03:18 AM

Hi,

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.

 

Alessandra 







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.