How to calculate library concentration for qPCR in pM?

[mbk0asis]

Hi, all!

I recently made illumina sequencing libraries, and I wanted to check them with qPCR.
But I wasn't sure how to prepare control libraries in pM.
I heard that I could get concentration in pM if use Bioanalyser or something, but we couldn't afford it.

So if any of you knows how to calculate the concentration of libraries in pM, please teach me!!!

[microgirl123]

Can you afford to use the KAPA quant kit - or just the standards from it?

[kcchan]

Illumina does sell PhiX control that you can use as a standard, but you can use any Illumina library as a qPCR control as long as you know the exact concentration of the sample. All you have to do is perform a serial dilution to create a standard curve and then calculate the concentration of your new library according to the standard.

[nhunkapiller]

Whatever Control you ultimately use, make sure that the fragment length is the same, or your quantitation will be off.

[TonyBrooks]

Whatever Control you ultimately use, make sure that the fragment length is the same, or your quantitation will be off.

I believe the Kapa kit contains a normalisation step to account for this during analysis (basically just divide the fluorescence values by the library size).

There is also a homebrew qPCR method for absolute quantification in this paper which uses TaqMan (FRET), not SYBR. http://www.ncbi.nlm.nih.gov/pubmed/21431778
There should be no normalisation needed for this one. But, of course, you will still need a standard.

A word of warning about the Illumina 10nM PhiX: There is always some batch-to-batch variation in the absolute quntification of Illumina PhiX. Generally, it's around 10pM, but can be >10% higher or lower. This is born out when looking at our cluster densities from various batches. The Kapa controls are always spot on as they normalise to previous batches by qPCR.
It should be good enough to get you started, but you may need to optimise a bit.

[share]

I agree with Tony. If you can't get kapa kits I suggest to use any of the library which were previously sequenced and who's cluster numbers are known. Before doing qpcr just have a look at kapa doccumentation to normalization for diffrent library sizes. http://www.kapabiosystems.com/public...lumina_TDS.pdf