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PCR product as standard curve template

real time pcr rt-pcr pcr standard curve

Best Answer doxorubicin, 25 July 2013 - 06:08 AM

That's what I do. You'll want to have some reference genes so that you can see "small" differences in total cDNA levels. Go to the full post


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#1 detriar

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Posted 22 July 2013 - 07:47 PM

Dear all,

I want to use my purified PCR product as template in standard curve absolute quantification. Are these steps seem correct to do:

1. I will use mcf7 cell-line cDNA as the template in conventional PCR, and the forward and reverse primers I'll use is the same with the qPCR primers. Then I'll excised the gel that contained target, and purified it using Roche Purification kit.

2. I'll quantify the concentration with nanodrop, and use the equation:
(X g/μl DNA / [pcr product length in basepairs x 660]) x 6.022 x 10^23 = Y molecules/μl

3. I'll dilute it in 10-fold serial dilutions 1:10, 1: 100,..... and use it as the standard curve template to establish copy number of the target.

The problem is, I'm not sure if these are the correct steps. My samples are frozen tissues, I extracted the RNA, and performed RT step. Then the cDNA will be used as the samples' template. If I performed the steps above, will I get the target RNA copy number or DNA copy number as a result?

On step number 1, can the same primers be used in the conventional PCR and then qPCR? As I'll use sybr green, will it affect the amplification?

And I'm not sure the equation on number 2 is correct to quantify molecules/ul. Can anyone suggests the other way to quantify it? Or if someone ever use PCR products as standard curves template?

Thanks.

Waiting for some help,

Cheers,
dee

#2 doxorubicin

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Posted 23 July 2013 - 01:50 AM

Your strategy seems reasonably good. Why not just report your data in nanograms per microliter? I definitely recommend making the standards using the cDNA from your frozen tissues rather than MCF7 cells. Also, use the same PCR master mix/enzyme to produce your standards as you will be using for the qPCR. This way you will be certain that you are comparing exactly the same PCR product when comparing standards to your samples.

#3 detriar

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Posted 23 July 2013 - 05:55 AM

Dear doxorubicin,

Thanks for your input, I really appreciate it :)

Yes, the PI of the research wants the final result as RNA copy number/ul, because we want to check the RNA copy number of the GOI before and after treatments. But since I get a little confused abt using the PCR fragment (which is dsDNA) as standards to get the samples result in RNA copy number/ul, I think I have to ask somewhere :).

So you suggested using the one of my cDNA samples as the template for conventional PCR rather than using the cell line? Can it be done? Though my samples are tumor tissues?

Many thanks,
dee

#4 doxorubicin

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Posted 23 July 2013 - 06:19 AM

I haven't thought about copy number before, but to me this seems logical: What you're really calculating is how many copies of the cDNA you have made in your RT reaction and comparing it to the double stranded cDNA standard. You can probably just use a calculator like this one once you know the number of ng and the size of your amplicon: http://cels.uri.edu/gsc/cndna.html

The reason I suggested using the exact same cDNA and qPCR master mix to generate the standards is that you want to avoid making a standard curve of a PCR product that isn't the same one you're looking at in your cells (SNPs, alternative splice products, etc.). Just take a small amount of the cDNA you made from a couple of your samples and do a test run to see if it gives a clean melting curve with your PCR primers. Then take the finished reaction and use a PCR purification kit to clean up this product, send a bit for sequencing, and make your standards with the rest.

#5 detriar

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Posted 25 July 2013 - 05:45 AM

Dear doxorubicin and all, once again thanks for the reply. I still get curious if there is any way so I can get the final result as RNA copy number/ul?

For now I think for now I'll get those steps above done and I'll tell you how it's going :)

I have this one question though, I've read it somewhere but I have to make sure, there's no need to nanodrop the cDNA conc, right? We just need to quantify the RNA conc. of all samples and take the same amount (e.g. 100 ng) and synthesize into cDNA, then use it as templates for qPCR?

#6 doxorubicin

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Posted 25 July 2013 - 06:08 AM   Best Answer

That's what I do. You'll want to have some reference genes so that you can see "small" differences in total cDNA levels.

#7 detriar

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Posted 25 July 2013 - 06:29 AM

Thanks a lot :)

Best Regards,
Dee





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