So I was running an RT-qPCR, but as you can see from the attachment some of my technical replicate triplets have CT values that vary by 1 (e.g. 26.16, 25.88, 25.14), leading to standard deviation greater than 0.5.
What to do then? Can I still keep the good results and re-run the problem ones on a new plate with the same housekeeping gene? I have made aliquots of the same samples and primers that I can use.
Also, any advice on how to prevent CT value variation in replicates. This is probably a pipetting error, right? As an inexperienced undergrad I spent 3 hours making this plate ;(
For sure you can re-run genes that was not okay in the previous plate using same primers and samples.
In my opinion there's 2 major sources of variation between technical replicates - pipetting error and bubbles in the well. If you have a bubble in the well it affects reading drastically so try to avoid them as much as possible.
Well, you're probably going to get better data if you repeat your plate.
One way to reduce the well-to-well variation of replicates is to make master mixes. In brief, you make up one tube that you then split between your three wells.
If you really want to be super precise (and like doing math), you make up a mix that has all the common ingredients for all your samples, split that into mixes for each template, then make the mix for each primer set. Be sure to include about 20% excess volume for each mix so you don't run short on the last sample.
Avoiding bubbles does help too. If you have a plate holder for a centrifuge you can give it a brief spin down before running it.
I agree, if you're not short on samples you for sure can repeat to get better results. But you also can keep a good ones.
Amy Klocko
So it's not recommended to remove the outlier in a triplicate and take the Ct mean of the remaining two?
It's not a recommended approach. It's better to repeat your experiment and use data from triplicate wells with similar Cts. Your current plate can give you a general idea of the trends. If you are planning to publish your findings they need to be improved in regularity between the identical wells.
You do not know why you have a problem. Thus you do not know if the results that appear good are correct or not. I would redo the entire plate.
Experience will make plating go faster. There is a learning curve. Laboratory methodology is also important. How consistently you use the pipettor determines the quality of the results. Spinning plates to remove bubbles is recommended. Being tired, rushing, or distracted increases variability.
Be clear on project goals and what is a replicate in your experiment. If the PCR is a tool, then make large batches. This will also enable the use of a repeating pipettor or multichannel pipettor. However, the issues with large batches are that you want to minimize waste, you want to mix the batch very carefully, and poor laboratory techniques will show up when you have to make a new batch. This is not so great if you make a mistake somewhere.
In making batches, make sure that all components can be stored together without reacting. Reactive components should only be mixed when you make the plate.
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