Hello. I am trying to compare relative expressions of a gene in several cell lines. I am using 20ng cDNA in 10 microliter qPCR reaction for each cell line. Actin b is the internal control i am using. When ctValue of actin is around 16th cycle, my targets are in around 33rd-34th cycle.
When i repeat the experiment several times, cell A has higher expression than cell B but in the next replicate, B has higher expression than A.
Is it not possible to compare very low expressing genes or does it have another cause?
Thank you very much
Hi Khorolgarav
Ct value at 33 - 34th cycle may resulted from low effective PCR.
Let try to improve your PCR first by add more cDNA. You can optimize the reaction by normal PCR and visualize it on gel electrophoresis for the better band intensity.
Hope it helps ^^.
A few things to check first: make sure you're not oversaturating your reaction with cDNA. 20ng isn't a huge amount (so I suspect not), but it's worth checking.
Basically, do the same reaction with varying amounts of cDNA, maybe from 1ng up to 50 or more, and see if your beta actin Cq values change accordingly.
If 20ng of cDNA gives you a Cq of 16, then 10ng should give a Cq of 17, 5ng a Cq of 18, 2.5ng a Cq of 19, and so on.
If you instead get a non-linear change in Cq, you might have saturation problems (running qPCRs with too much cDNA tends to inhibit the PCR reaction, either via primer chelation or cDNA synthesis buffer effects), so make sure your cDNA amount falls within the linear range of your assay.
Having established that, I would be tempted to suggest that your gene of interest is simply not expressed to any biologically-relevant level. Cq values of 16 for beta-actin are not unreasonable (and represent something in the order of a million or so templates per well): if under the exact same conditions your GOI gives Cq values of 33-34 (within the range that corresponds to fewer than ten template molecules per well), it might as well not be there. It's either expressed at a biologically relevant level in one cell out of thousands, or it's just background noise.
If you still think this data is real, however, you can improve the quality of your data by simply using more replicates. Very low expression levels lead to stochastic partitioning effects: with fewer than ten template molecules per microlitre, it's not unusual for any given microlitre to contain twenty, five, two, seven, or even zero templates.
You will get highly variable Cq values simply because the amount per well varies readily when there is so little template present.
Performing reactions in triplicate is thus insufficient to get quantitative data, while 5 or 6 replicates (averaged) might well be representative.
Thank you very much, dear researchers. The answers are very important for me.
Agree with the suggestions given above. Two things you could also do. 1. Using a cell line or tissue known with modest to high expression of your gene of interest as a positive control; 2. designing another (or two) pair of primer (probe) to run the samples you already have just in case the efficiency of the primers you used is too low. Good luck with your experiment.
- Yes this can happen with low copy number genes; that is genes with Ct values > 30
- I have never really figured out a solution to this problem apart from pre amplification of your cDNA: I can send you details if you wish
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