question about qRT-PCR preparation
Posted 06 July 2010 - 11:31 AM
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!
Posted 07 July 2010 - 05:35 AM
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.
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