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Old 12-06-2012, 07:29 PM   #1
cmuletz
Junior Member
 
Location: Washington, DC

Join Date: Jul 2012
Posts: 9
Default Qubit versus qPCR

Hello,

My question relates to the use of Qubit to qPCR for quantifying your amplicon samples to equimolar ratios -- we have both a Qubit/dsDNA HS Assay Kit and qPCR/ Kapa kit in our lab to quantify 16s rRNA amplicon samples with fusion primers. I will pool ~30 samples together. From looking at the previous threads people recommend qPCR and that is the same with the folks in my lab over Qubit and Qubit over nanodrop. However, most microbiome papers I have found people are using Qubit to quantify before pooling. If most people are doing this then...?

I'm asking for accuracy and time efficiency.

1) How do you feel about Qubit versus qPCR?

2) For qPCR are most people doing absolute qPCR over relative? -- I'm not sure if you need the Kapa Kit to do relative qPCR (you would still need to quantify some samples at some point though).

--relative seems faster with the accuracy -- you have the qPCR results but you don't need to go through the dilutions and replicates to do the absolute qPCR. For absolute qPCR I've been told that you need to do a few dilutions and at least 2 replicates and you need the standards so you can really only quantify 10 samples at a time. I realize with relative you would still need the reps.

--for my 30 samples that is 3 runs on the machine for absolute versus 1 for relative. In the end I will be doing 4 more pools like this.

Any folks with experience your comments would be greatly appreciated.

Thanks!

Carly
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