using PCR product as standard template

Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum

Moderators: r.rosati, Suzanne

using PCR product as standard template

Postby Aidanmc » May 25 2010 10:56 am

Hi All,

I want to use purified PCR product as my standard template in order to perform absolute quantification, here is my plan:

1. I will use the FWD and REV primers that I plan to use in future qPCR to amplify my product (GAPDH) using cDNA as the template, I will do this on a regular endpoint PCR cycler.

2. I will run the product on an agarose gel, cut the band and purify using a promega wizard column (should i elute in nuclease free H20 or T.E? which will the product be more stable for??)

3. i will quantify the purified product on the nanodrop

4. I will use the concentration along with the product sequence to work out copy number per ul of my sample (I am unsure of how to do this, what equation do I use?)

5. I will then prepare 10fold serial dilutions of my purified product and use this as my standard for absolute qPCR of my samples to establish copy GAPDH copy number.

Is this ok?

can you guys give me some guidance with point 4?

thanks

Aidan
Last edited by Aidanmc on May 27 2010 7:19 am, edited 1 time in total.
Aidanmc
supertech
supertech
 
Posts: 70
Joined: Jun 11 2008 11:16 pm
Location: SYDNEY

Re: using PCR product as standard template

Postby JMG » May 25 2010 9:00 pm

Hi Aidanmc

According to a place called “IDT” (Integrated DNA Technologies), nuclease-free TE pH 8.0 is best to store your amplicon
in (since DNA is more ‘stable’ at pH 8.0)

For your 'point 4':
It will be your calculation of the Molar extinction coefficient of your PCR product that is key ...

You will either need to calculate the molar extinction coefficient of your GAPDH product based on its sequence, using the dNTP pH 8 values (if measuring in TE pH 8) for each base (ABI values shown below) and add up all values to come up with the final sum value for an estimate, or, by using the “nearest neighbor” method which takes base order/stacking into account...

ABI values for deoxyribonucleotides (dNTPs) at pH 8.0:
dA = 15200 M-1cm-1
dC = 7050 M-1cm-1
dG = 12010 M-1cm-1
dT = 8400 M-1cm-1

So, if your sequence is ATTGCT... you would add 15200 + 8400 + 8400 + 12010 + 7050 + 8400 ... and so on for the sense strand, then do the same for the anti-sense strand until you get the final summed value for both strands - which would represent your molar extinction coefficient for your dsDNA GA3PDH PCR amplicon product sequence.

See more details on this calculation on p. 11 of the following:
http://cdn.idtdna.com/support/technical ... torage.pdf

Aside: [And, remember, to the qPCR, a double-stranded product = 2 copies of target. So, whatever you are assessing (e.g. gDNA vs. transcriptome), be sure you remember this. gDNA is dsDNA, and transcriptome is ssRNA (thus single-stranded cDNA starting as the first strand)... in general. So, 1 copy of amplicon on its standard curve = 1 copy of gDNA on its standard curve since both go into the qPCR as dsDNA species. But, if using a dsDNA plasmid to help calculate copies of a transcriptomic target mRNA/cDNA, then one must remember that 1 copy of plasmid is actually 2 copies of target when comparing it to a transcriptomic target …]

Anyway:
Once you have computed the extinction coefficient for your GA3PDH amplicon sequence, (let’s say you calculate it to be 2532360 L/mol*cm. And you diluted it in TE pH 8.0, and, let’s say, at a 1:10 dilution of your amplicon (after the NanoDrop has been blanked using TE pH 8.0) you get an A260nm absorbance reading of 6.3309 on a NanoDrop. Using the Beer-Lambert equation, A = ebc, we arrange it to the expression:
(10^6)*A260nm*dilution/eb = c (in uM): so we calculate your undiluted amplicon solution concentration (in uM) to be: (10^6) * 6.3309 *10 / (2532360(M^-1cm^-1) * 1 cm) = 25 uM

So, if we find by NanoDrop that you have a 25 uM solution of your purified, dsPCR product:
since we know that uM = picomoles/ul ... and we also know that Avagadro’s number = 6.02214179 x 10^23,
then, since 25 uM = 25 picomoles/ul, and 25 picomoles/ul = 2.5 X 10^-11 moles/ul,
we can calculate that 2.5 X 10^-11 moles x 6.02214179 x 10^23 = # of dsGA3PDH amplicons/ul =
1.50553545 x 10^13 dsGA3PDH amplicons/ul (again "ds" here connotes "double-stranded")

This means that your 25 uM solution of purified dsGA3PDH amplicon in TE pH 8.0 contains 1.50553545 x 10^13 double-stranded GA3PDH amplicons in each ul. You can do your serial dilution calculations for your standard curve from here. And, the amplicon should work nicely as the material for a qPCR standard curve substance (as opposed to super-coiled plasmid DNA).

See if these calculations help you at all ... let me know if I lost you along the way here or not.

As you will find, it all comes down to how well you calculate the extinction coefficient of the amplicon is question
(e.g. which equation or equations you decide to believe, and use for that, is crucial ... everyone may do that
slightly differently -- perhaps Maniatis and Fritsch have a solid, unchanging idea on this ... e.g. Maniatais,
T., Fritsch, E. F., and Sambrook, J. (1982) Molecular. Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY ...) -- an exact approach (used by everyone everywhere) is a hard to dig up...
but, it would seem that the nearest neighbor method is probably the way to go. A good calculation of the molar
extinction coefficient calculation is key.

And always remember, the inverse of the molar extinction coefficient tells you what Molarity your solution
would be if your A260nm reading is exactly 1.0


I find my way of doing these series of calculations fairly logical ... it is disconcerting that this seemingly basic maneuver
is so hard to find published in explicit/succinct terms isn't it?

JMG
Last edited by JMG on Jun 04 2010 3:00 am, edited 1 time in total.
JMG
PI of Posters
PI of Posters
 
Posts: 620
Joined: Dec 19 2008 3:13 am

Re: using PCR product as standard template

Postby Aidanmc » May 26 2010 7:25 am

Hi JMG,

Once again, a very extensive and useful answer, really appreciate it.

I have calculated my extinction coefficient to be: 20 382 320 (my PCR product is 966bp long)

Today i performed my PCR and purified the gel slice on a wizard column and eluted in 50ul T.E pH 8.

I then blanked my nanodrop with this T.E and loaded 1.5ul (undiluted) of my purified product:

76ng/ul
260nm: 1.52
260/280: 1.92
260/230: 2.05

So,

(10^6)*A260nm*dilution/eb = c (in uM)
((10^6) * 1.52) / 20 382 320 = 0.0745744351

So I have a 0.0745744351 uM solution of my dsPCR product:


0.0745744351uM = 0.0745744351 picomoles/ul = 0.00745744351 X 10^-11 moles/ul,
0.00745744351 X 10^-11 moles x 6.02214179 x 10^23 = 0.00745744351 X (10^(-11)) x 6.02214179 x (10^23) = 4.49097822 × 10^10

So my purified product has 4.49097822 × 10^10 ds GAPDH DNA amplicons in each ul


does this look correct?
Aidanmc
supertech
supertech
 
Posts: 70
Joined: Jun 11 2008 11:16 pm
Location: SYDNEY

Re: using PCR product as standard template

Postby JMG » May 27 2010 2:36 am

That's exactly what I would calculate~
To me it is correct -- but, I am just one guy --
if we could cross confirm with Suzanne or mchlbrmn
or Ampl^nth or researchwoman or Sing09 here - I would
feel much better about this...

JMG
Last edited by JMG on May 27 2010 12:46 pm, edited 1 time in total.
JMG
PI of Posters
PI of Posters
 
Posts: 620
Joined: Dec 19 2008 3:13 am

Re: using PCR product as standard template

Postby Aidanmc » May 27 2010 7:18 am

nice one, it is strange that there is no publication for such a calculation, maybe you should publish your method JMG?



so I have ~45 billion copies per ul, I want my std curve top point to be 100K, got alot of diluting to do!
Aidanmc
supertech
supertech
 
Posts: 70
Joined: Jun 11 2008 11:16 pm
Location: SYDNEY

Re: using PCR product as standard template

Postby JMG » May 27 2010 1:19 pm

A lot of diluting to do - but the main thing is to achieve
a certain desired series of "copies per reaction" of course...

E.g. 10,000,000 copies per reaction down to 1 copy
per reaction would give you a nice dynamic range
of 7 orders of magnitude.

But, your amplicon standard will often have a different efficiency of
amplification than your samples will (for the same GA3PDH target
using the same primers/probe). So, be sure to mathematically
reconcile the two with one another so your amplicon standard curve
can more truthfully tell you how many copies of GA3PDH there are
in each of your biological samples.

e.g. For GA3PDH target in your amplicon curve as compared to
GA3PDH target in your biological samples:

Amplicon:
initial GA3PDH Copies in the reaction = (Xo)a
exponential amplification value = EAMPa = 1.92
b = y intercept of standard curve; which tells you the Cq of "1 copy" = 42.3 = ba

Biological sample:
initial GA3PDH Copies in the reaction = (Xo)s
exponential amplification value = EAMPs = 1.87

Using the above info, we first need to find out what "b" would be for GA3PDH
in the biological sample at its particular GA3PDH efficiency (having run a standard
curve for GA3PDH using the biological samples, we know GA3PDH went off at 87% efficiency):

So, if b = 42.3 for the purified amplicon standard curve (which goes off at an
exponential value of 1.92 or 92% efficiency), this means that 1 single
strand of GA3PDH (technically 1 copy) shows up (crosses threshold),
can be detected as signal above background at 42.3 cycles.

But, what does that mean in terms of the biological sample's GA3PDH?

To apprehend "b" of 1 copy for the biological samples (let's call it "bs") we can
use the following transformation:

bs = ba*log(EAMPa)/log(EAMPs)

Thus bs = 42.3*log(1.92)/log(1.87) = 44.08317817

Then, when we use the equation: (Xo)s = 10^((Cq-bs)/m) for
assessing GA3PDH in biological samples, the resulting values will be in terms of
copies per reaction since "bs" in the equation literally means the Cq at which
1 copy (1 strand) of GA3PDH would appear in your biological samples given their
particular GA3PDH amplification efficiency allowed by using the primers and/or
primers/probe and master mix that you do.

The single-stranded versus double stranded issue here (notice how it still lurks and haunts even at
this point) -- always be aware of how you reason with that. To me, "one copy" does not automatically
imply "two strands", and to qPCR and PCR in general, 2 strands vs. 1 strand constitutes a big difference:
e.g. twice the amount of product is made in the end when a double-stranded species enters the reaction as
opposed to single stranded species. A double-stranded target melts in two first, and presents "2 copies" to the
PCR, while a single strand presents just "1 copy" to the PCR. E.g. (and I tend to repeat myself a lot on this point
here): a dsDNA plasmid with a single target insert contains 2 copies of that target since, when the plasmid melts
apart during the first stage of PCR, it opens to expose a sense and an anti-sense target sequence - one to which the
Fwd primer can bind, the other to which the Rev primer can bind. The net PCR result is 4 copies of target sequence (not
2 (which would result if only 1 initial strand of target template presented itself to the PCR).
Last edited by JMG on Jan 20 2012 12:18 am, edited 1 time in total.
JMG
PI of Posters
PI of Posters
 
Posts: 620
Joined: Dec 19 2008 3:13 am

Re: using PCR product as standard template

Postby rosemac » Oct 21 2011 8:53 am

Hi! I am a qPCR neophyte and I am having trouble with the standard curve. I cannot make it to perfect R^2 =0.99. It's been from 0.95 down to 0.71 after several attempts. I tried reducing the primer concentration from 500nM down to 100nM but the curve gets worse everytime.I used the cDNA as a template and made 1:10 serial dilutions to make a 5 point standard curve plus NTC. I based the gene copy numbers on the primers as a reference value to be 10^16 . Although the curve is not perfect the calculated gene copy numbers are similar after the run. I suspect that my pipetting techniques affect the efficiency.

I have 2 concerns: (1) I am planning to use my previously prepared PCR product as a standard template for my next qPCR run, but not purified from the agarose gel. Please let me know if I can use the amplicon directly from the tube and dilute it 10 folds in RNAse-free water. (2) If I get a perfect curve, can i just import it everytime I run my samples, instead of running the standard curve with the samples to save on master mix reagents?

I appreciate any response.

:) rosemac
rosemac
newcomer
newcomer
 
Posts: 1
Joined: Oct 21 2011 8:13 am

Re: using PCR product as standard template

Postby Albal » Sep 13 2017 7:16 am

Hi,
I am a newbe in qPCR. I want to use my end point PCR product as standards in qPCR. I am trying to amplify the gel purified PCR product to amplify with the same primers and I am getting only a smear. I do not know what I am doing wrong. If my standard PCR is failing to amplify, how will it work as standard in qPCR?

Your guidance an support is highly appreciated.

Thanks
Albal
Albal
newcomer
newcomer
 
Posts: 1
Joined: Sep 13 2017 7:08 am

Re: using PCR product as standard template

Postby JMG » Sep 13 2017 4:02 pm

I would suggest doing a serial 1:2 dilution of your PCR product and try to amplify it
at different dilutions. You may be inhibiting the PCR by adding too much cDNA
to the reaction...
I will not be able to respond further on these topics as my job has been eliminated.
Best to you~
JMG
JMG
PI of Posters
PI of Posters
 
Posts: 620
Joined: Dec 19 2008 3:13 am


Return to Real-Time qPCR/qRT-PCR Methods

Who is online

Users browsing this forum: No registered users and 4 guests

cron