I seeded PC3 cells in 2 T75 cm2 flasks (8 x 106 cells/T75). After incubation overnight with 5% FBS-Depleted EVs medium, I isolated exosomes from cell culture with a Qiagen kit and I performed a reverse transcriptase. Subsequently, I did a qRT-PCR with some genes and below you can see CT where they appeared:
Exogenous controls for Isolation: UniSp2 (CT 19,60), UniSp4 (CT 26,89).
Exogenous controls for Reverse Transcriptase: UniSp6 (CT 20,93), cel-miR-39-3p (CT 28,44).
miR-103-3p as a stable reference gene (CT 25,01).
Results show a good isolation and reverse transcriptase procedure, but I’m not sure if I’d have enough cells to analyse a microRNA panel (miRCURY LNA miRNA miRNome PCR Panels – Human panel I + II) with the housekeeping’s amplification results.
Can anybody give a hand or advise about it, so I can go ahead with the Panels.
Thanks in advance
Your housekeeping is the right level to be sensitive to differences. Overall, you want to have your Cq between 18 and 28 (actually Ct is the old denomination, now it is cycle of quantification, Cq).
In this range, usually microRNA amplication (qPCR effisciency) is quite around fold 2 by cycle, which is not the case below 18 and above 28.
I would try to quantify of one the microRNAs from the panel and see its levels. It can very well have lower Cq than your stable reference microRNA because, on the contrary to RNU6 or GAPDH, there is no reason for which your reference microRNA has to be highly expressed as housekeeping genes usually are. So your panel could possibly be at lower Cq.
By monitoring one of the microRNAs of your panel, you could get an overview. If it get above Cq 30, then you might want to get more cDNA.
Concerning your exogenous spike-in controls, those are important if you are comparing multiple groups.
So it might depend on how many groups you have. If you are worried some of them might have too high Cq, it is usually a good practice to make a pool of all your groups, try your panel on the pool which will give you the "overall" levels of the microRNAs you are monitoring.
Hope it helps !
Cheers !
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