No significant amplification in real time

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gin
gin's picture
No significant amplification in real time

Hi
I am trying to figure out the expression profile of a bacterial gene in different condition using real time PCR. I have two doubts reagarding the two of my observations as i am handling the real time system for the first time. Firstly i am getting a conc. of primers (For. and Back.) - 10 nM each in standardization, which I guess is slightly lower of what one suppose to get in these analysis. On increasing the concenteration even to 25 nM the self amplification and dissociation attributes do not remain consistent. Though they are very much consistent in 10 nM conc. of Fw and Rv.
Secondly, I am not able to get significant amplification of the cDNA from my samples with 10 nM conc. of primers. It is happening in both the cases, as when i put 5 microgram/50 microlotre RNA for cDNA conversion as well as 20 microgm/50 microL (which is 4 times higher than recommended). I doubt that the cDNA might contain too less of my target transcript or its time to order new sets of primers.

Jason King
Jason King's picture
It sounds like you are unsure

It sounds like you are unsure whether it is the cDNA synthesis or the PCR reaction that is not working. Most people I know do cDNA synthesis reactions using 1 µg total RNA in a 20µL reaction volume. However, I have also done it using down to 100ng and it still works (AB cDNA synthesis kit). Other suppliers have kits that allow you to get sensitivity at 1ng total RNA, so the amount of starting material shouldn't be a problem. If your cDNA is OK, you should be able to use it to amplify a house-keeping transcript. If you can do this then you need to look at your PCR conditions.
What kind of real time PCR are you doing? Have you also tested your primers in a standard PCR reaction?

Ivan Delgado
Ivan Delgado's picture
 

 
Hi gin,
Here are my two cents. As far as the concentrations of the primers is concerned, 10 nM is very low. The lowest concentration of primers suggested is 50 nM. I routinely run assays using 500 nM, and 1 uM (micro molar) works perfectly fine.
As parvoman points out the standard RT reaction recommends 1 ug of total RNA. That should be way more than enough. If you are using 5 to 20 ug you are likely introducing inhibitors into your RT reaction that quite possibly stop any cDNA from being synthesized.