I am using a 24 well plate and the cells are too few after 24hrs and I am concerned about my RNA yield. I would need to proceed with RT PCR for gene expression studies. I am using 0.5 micromolar camptothecin in 10 exp. 5 cells.
You can perform a time course of 24 - 48 and 72h or even more. If your siRNA is targeted against a protein you should also show that it has been knocked down as well by western blot. Most reviewers will ask you for it..
Do you need to perform your experiments with low amount of cells? If not, try to seed more cells. Otherwise you can try to look for camtothecin concentration modulation, Maybe the conc is to high (and it's toxic so cells don't proliferate and die), or look for specific RNA extraction kit for low amount (in the past I used cell to ct kit to extract RNA and perform RTPCR from 10 cells laser captured).
As pointed by Abdelhalim its better to extract after 24-48 h transfection
When working with an siRNA set-up, I always extract RNA 48-72h post transfection.
I've used Qiagen HiPerfect reagent and a fluorescent control siRNA along with test siRNAs. Although the best time for fluorescent checking is 6-24 hrs post transfection, silencing seems to be optimum after 48-72 hrs, but I guess this would vary depending on the targeted gene.
use Cells to Ct kit - if you just plan to do qRT-PCR downstream. no isolation of RNA, no sample loss, fast and simple, you just work with a crude cell lysate. great for a very wide range: from 10 to 100,000 cells
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