Hi Everyone,
I have been working with a FAC sorted population of embryonic brain cells. I typically get between 50-90% viability following the sort but only 12 000 - 20 000 cells. I have been tired a few different methods for RNA extraction including the RNeasy Micro Kit and and Trizol-Chloroform Extraction. I then assess the concentration and quality of my RNA using a Tape station assay. According to my results the RIN numbers are above 9.0 but the RNA concentration is very very low - ranging from 0.5 to at most 3ng/ul.
Does anyone have experience extraction RNA from low number of cells between 10 to 20k, and successfully used it for downstream applications like producing cDNA and running qPCR?
I have spent a lot of time optimizing the FACs sort, and am getting decent viability but now am running into trouble with RNA extraction.
Any input on those who use low cell numbers, their typical concentration achieved following RNA extraction and which method they use would be greatly appreciated it!
Hello Faizan
I have worked with a FAC sorted population of luteal cells. I got between 10 000 - 50 000 cells, I used Trizol to extract RNA and Ribogreen RNA assay to measure RNA extraction, I got 10ng from 10k cells and it worked well with Qpcr
Regards
Hi Faizan, How much trizol did you use? I think you should use trizol-chloroform extraction method with high amount trizol. Alternatively, you may use sonicator for cells in trizol. Try to wait in trizol more than 15 min. Good luck.
Hi
Two general points from my experience. First I would recheck that you are not losing cells due to surface adhesion. We were told to use special facs tubes (I guess Polypropylen but I am not completely sure pls. Recheck) and we were asked to "rinse" the tubes with Facs buffer containing BSA prior to sorting.
In general you can assume to get between 10-30 ng of total RNA per 1000 cells. And we are routinely doing cDNA synthesis with 1ng to total RNA this works fine, of course you might loose extremely low abundant targets
Good luck Sven
Hi Faizan Malik ,
Were you able to optimize RT-PCR on the low amounts of RNA you were getting?
If so, could you please let me know what worked for you?
Thanks!
Morgan
Hi Sven Reister
What enyzme/protocol are you using for cDNA synthesis?
Thanks,
Morgan
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