question about qRT-PCR preparation - (Jul/06/2010 )

I use ABI one step qRT-PCR system. I am hesitating between two ways of setting up the final reaction mixture.

1) prepare a tube of mixture for triplicate reaction for each sample. The final step is to add 20ul final reaction mixture into the 384-well plate;

2) prepare a master mixture for all reactions; add RNA samples (in triplicate) into the 384-well plate. The final step is to add the aliquot of master mixture into the wells which already have RNA samples in them.

I felt the second way is easier than the first way, especially when I have many RNA samples and quite a few genes of interest. But with RNA samples in the 384-well plate (= open to the air) all the time until addition of the master mixture, I worry about any possible contamination/degradation.

Which way do you guys use? I'd appreciate any suggestions on this issue! Thanks in advance!

One master mix for all samples, mix, spin, aliquot into eppendorf tubes for triplicates, then add RNA into each tube for a triplicate, mix, spin and pipette 20 ul into the plate. That's how we do it. You only have to add excess volume to each of the triplicate and once for the whole mix of them, for the pipetting error.

This way you have a mix for all the samples, thus comparable, and evenly pipetted template.

If you use the approach #2, you will have higher std deviation then using approach #1, because you pipette more smaller amounts of your sample. Anyway if using #2, you should pipette the mastermix first, then add RNA to each well, you would expose your RNA less.


By the way, 384-well plate is a job for a robot, doing so much samples is exhausting for a human and you can make pipetting mistakes. Also the danger of cross-contamination is higher.