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Optimization of real time-PCR - where should I start from?


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#1 Nephrite

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Posted 17 April 2018 - 09:11 PM

Hello!

Here I have 36-well Rotor gene 6000 cycler and I want to do relative quantitation via two step RT-qPCR - I mean, first I make RT reaction, and then use the product for qPCR.

It works without ROX.

I have some brief manual of the cycler but it doesn`t say much about how to analyze my results and how to optimize the reaction.

I downloaded several manuals, I continue to read, but I have some questions:

 

1. Should I prepare standard curves for any of the primer pairs with different cDNA concentrations? 

 

2. I read that I have to optimize the concentration of the primers for to avoid dimerization - can I do this by running several NTCs with different dilution of primers or I have to sacrifice some of my RT product? I ask this silly question because the NTCs of some primers are fine, but others have some peaks.

 

3. What is better - to have separate annealing and elongation step or to unite them? The protocol of the kit includes separate steps - is that ok?


Edited by Nephrite, 17 April 2018 - 09:12 PM.


#2 bob1

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Posted 18 April 2018 - 06:18 AM

1) As you specified relative quantitation you don't need to do standard curves; those would be for absolute quantitation.

 

2)The reactions might be different in the presence of the rt product, as in the absence of target you can get some primer-dimerization. I would see how it goes with no template and then test with template.

 

3)Follow the kit.



#3 Nephrite

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Posted 23 April 2018 - 02:35 AM

Hello, bob1.

Thank you for helping me again.

I resolved my problems with samples, primers, etc., and have some progress.

The program of the real time PCR cycler however wants me to specify some standards in order to determine the threshold line automatically.

Otherwise I have to determine it manually in order to gain the Ct values.

That`s why I made the question about the standard curve.

I am not sure what is the correct way to determine the threshold line - automatically with a starndard curve or manually for each assay?

I would also like to achieve some more professional level in real time PCR.

I assume that the manuals do not say everything.

Is it worthy to pass some on-line training?






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