Hello,
I'm new to RNA extraction and RT-qPCR. At the moment, I'm doing a lot of RNA extraction. My first initial result was actually good with high concentrations of RNA and pure. This stops when I start to move into my real project.
So my supervisor has already seeded and preserve all the cell lysate in Trizol in 24-well plates. My job is first to extract all the RNA from these plates. However, no matter how careful I am, the level of RNA in the final 30ul is always really low (ranging from 5 ng/ul to max 40 ng/ul, the purity is fine, above 2 most of the time, however). I assume this is because we didn't have enough of the lysate per well (aka the wells of the 24-well plate doesn't have much lysate because it's too small) at the beginning, thus result in low level of RNA.
We want to do RT-qPCR, do you think with this level of RNA, the qPCR still works? Maybe it will take more cycles, but will the result itself be reliable? Any suggestion? We also kinda in shortage with reagents too so it's kinda not ideal to use too much reagent.
Thank you very much
P/S: Also, we have a lot of samples, about 25 or so 24-well plates, I have extracted most of them (in the past 2 and half weeks or so). So reseeding the cells and redo those plates isn't really a good idea though.
15-40ng will sure work. But for very low concentration you can choose a high sensitive Sybergreen
Maynak Chakraborty: Oh really? But I thought you need at least 1 ug of RNA (in a 20ul reaction well)? Maximum here I can only use 11ul or the RNA, which makes 40ng/ul no where near it.
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