Pico Green vs Real Time PCR

[crndy52]

Does anyone have any experience with quantifying 454 libraries using qPCR (Real Time) instead of Pico Green. Our recent attempts have shown at least a 10 fold difference between the two methods using the same library sample.

[n2onme]

I have some experience with the pico quantification and qPCR quantification on 454 libraries. I have seen that the qPCR quants are normally lower than the pico quants. This sounds right to me due to qPCR measuring actual dna that will be amplified with the 454 primers, while the pico is giving you a quantification of all dsDNA.

[clarancer]

I routinely use qPCR (KappaBiosciences) to quantify my 454 libraries and it works very well. As well as running the standards in the kit I also run a library that I have taken through an emPCR titration and 454 run and that I know works well (i.e good enrichment and high proportion of wells passing filter). I have had a couple of occasions when this control library hasn't given the quantification that I expect so I have repeated the qPCR until it does and the values I get for my unknown libraries then work well in emPCR. Since we switched to this method we routinely get wells passing filter in the high 60 to low 70% even with bead enrichment levels approaching 20%.

One word of caution though is that be wary of the size span of your library. The standards that the qPCR kit supplies look to be around 450bp and if you have a lot of fragments in your library that are considerably higher than that then they won't amplify as efficiently during the qPCR and you will get a lower than actual quantification. My libraries tend to have a size span between 450bp and 1kbp so are affected by this but haven run a few samples through I have a good handle on what will give me a 12% enrichment and use this value and that gives me good leeway so that I always generate sufficient beads from a full scale emPCR for a 454 run.

Clarancer

[crndy52]

Thanks for your very helpful comments. It seems that qPCR is the way to go. Today I ran across the paper at the link below paper that deals with this subject in detail. It is entitled "Titration-free massively parallel pyrosequencing using trace amounts of starting material".

http://nar.oxfordjournals.org/content/38/13/e137.full

[crndy52]

qPCR Method

Clarancer: Do you use the SYBR Green method in your qPCR? I have tried that and gotten mixed results. The paper I linked to above uses a custom Taqman MGB-probe that is specific to the adapters used in the Roche library prep. After reviewing the results these people got, I am seriously considering giving this method a try. Has anyone else tried this unique Titration-Free method?

[clarancer]

I've been using SYBR green and it has been fine. I dilute my libraries 1 in 500, 1 in 1000 and 1 in 2000 and then quantify against the standard and average out. I know that some people use the FAM label to get a 'rough' quantification and then use this value to dilute to 1e7 then use qPCR.

I did wonder whether the FAM label maybe a problem with the SYBR green detection and was tempted to buy some unlabelled adapters from IDT but given the results I've been getting I can't see the point.

The other thing that I sometimes do if I get an unexpected result by qPCR is to run the qPCR products out on an agarose gel (2% E- gel) against a 100bp ladder and look to see what the size span is. If a sample is high GC or AT then this can skew the size seen on the Bioanalyser. On the odd occasion this has shown my library to be smaller than I expect and as such there will be more molecules per ul and this causes higher than expected enrichment levels on emPCR.

Hope that helps

Clarancer