We tried to do qPCR quantification of some Illumina paired-end mRNA seq libraries (+-325bp in length) using the following primers:
qPCR primer 1.1 5’ AATGATACGGCGACCACCGAGAT 3’
qPCR primer 2.1 5’ CAAGCAGAAGACGGCATACGA 3’
The primers anneal to the 5'ends of the adapters and should thus amplify the entire fragment (the qPCR product size should be exactly the same as the original library product)
After doing a serial dilution of a library (with known cluster numbers) we performed qPCR on a Roche LC480.
We used several other libraries (with known sizes) as the unknowns. The standard curve generated was good with PCR efficiency of 93% and very low error. All of the amplifications looked good (Cp<15) and no template controls produced no product at all after 45 cycles.
So on first impression, it seemed that the qPCR worked well - that is until we ran the products on agarose to see the product sizes. It turned out that the products were quite mixed but the prominent product which should have been the same size as the library turned out to be about 200bp larger than the library products.
Pictures below show original library product and qPCR product (arrow). Other libraries (unmarked lanes) also feature the same pattern - product +-200bp larger than the library band.
Any suggestions as to why the qPCR product was so much larger?
Anyone out there doing routine qPCR quant of libraries - your input would be much appreciated!
qPCR primer 1.1 5’ AATGATACGGCGACCACCGAGAT 3’
qPCR primer 2.1 5’ CAAGCAGAAGACGGCATACGA 3’
The primers anneal to the 5'ends of the adapters and should thus amplify the entire fragment (the qPCR product size should be exactly the same as the original library product)
After doing a serial dilution of a library (with known cluster numbers) we performed qPCR on a Roche LC480.
We used several other libraries (with known sizes) as the unknowns. The standard curve generated was good with PCR efficiency of 93% and very low error. All of the amplifications looked good (Cp<15) and no template controls produced no product at all after 45 cycles.
So on first impression, it seemed that the qPCR worked well - that is until we ran the products on agarose to see the product sizes. It turned out that the products were quite mixed but the prominent product which should have been the same size as the library turned out to be about 200bp larger than the library products.
Pictures below show original library product and qPCR product (arrow). Other libraries (unmarked lanes) also feature the same pattern - product +-200bp larger than the library band.
Any suggestions as to why the qPCR product was so much larger?
Anyone out there doing routine qPCR quant of libraries - your input would be much appreciated!
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