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Old 07-01-2016, 07:29 AM   #6
tmm343
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Location: New York

Join Date: Dec 2012
Posts: 14
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Hi All,

We normally qPCR but for these 16S and 18S libraries we have found Qubit to be more than sufficient and have been using it successfully and consistently and just recently ran into this issue and seeing this discrepancy.

How we came across the issue was using the Quanit BR reagents and a plate reader for the individual libraries and doing a final QC with the Qubit on the pooled sample as a double check. The Qubit assay initially used was the HS. While the concentration and pooling was done with the Quantit BR to a 4 nM pool the Qubit HS told us the pool was at 2 nM. We decided to use the 2 nM concentration and overclustered our run leading us to believe the BR assay was initially correct. We decided to go back and do this comparison and found the variation to be between the BR and HS assays which is showing using both Qubit and Quantit reagents. It becomes even more complicated the for 16S and 18S samples we see this discrepancy but seems as if the HS is accurate for the 18S samples and BR is accurate for the 16S samples.

@microgirl123 we are using fragment size to do the conversion to nM. The sizes are pretty consistent between all the samples.
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