Hello all. I have recently been trying to isolate total RNA after transfection of miRNA mimics and antisense inhibitors in mammalian cells. However, the RNA isolated through the trizol method attains very less yield, along with poor RNA quality sometimes.
Is this a common observation? Does it get resolved by kit based RNA extraction?
I did a similar experiment in K562 cells using Ribopure kit (Invitrogen)
Hi Varun, I did the transfection of miR inhibitor in NRK-52E cells. But in my first try to estimate the gene expression of a given miR in these cells, as I never did RNA extraction before that time in adherent cells, just in tissue samples, I didn’t have much experience in how I should use the scrapper to get the cells from the culture plate, so when I quantified the RNA extracted from that cells I quantified in nanodrop a very low quantity of total RNA extracted by trizol method, if I remember it was around 100 ng with poor quality. Then, I repeated the culture and at that time I used the scrapper more vigorously to scratch the bottom of the plate, besides that, I passed the scrapper more times at the bottom of the plate, and in practically all surface of the plate, differently that I did in the first experiment. In this last experiment, the quantity of total RNA extracted was higher (~250 ng/uL) and had good quality, but even that the quantity was low for my purpose. Another thing that I did to obtain a greater quantity was liophilized the total RNA and after that resuspended the sample in a lower volume to increase the concentration. To guarantee that the result obtained was right, I repeated the experiment and got the same results.
I hope that it helps you.
Pedro
Varun .K , please tell to us if doing what I suggested to you worked for your cell type.
Best
Pedro
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