I need to know more or less the yield in ng/µl from a million of cells
Hello Raúl,
see: http://www.wpi.edu/Pubs/ETD/Available/etd-0424102-141341/unrestricted/warke.pdf page 27
"Typically one million cells (1 × 10 6 cells) produced 10 µg of total RNA."
I tried to isolate 1 million cells using RNA isolation KIT and one million with TRIZOL method. Trizol was way better and it gave me approximately 50 µg of total RNA, whereas with Kit it was 10µg of total RNA
My idea is work with Trizol method in order to preserve small RNAs, then your news are better Kanika. Thanks a lot.
you can find several indications concerning RNA extraction from cells, anyway (as you can understand based on the answers already written) the real yield depends on the extraction protocol/kit/technique you adopt.
When I have extracted RNA from HUVEC, I got approximately 10 ug total from a confluent 35mm dish (which should be approximately 1 x10^6 cells) using the Trizol method. Though this was several years ago when I had less experience using Trizol and it will depend on the donor and what you consider "confluent" since I did not do a cell count, so this may be on the low end of yield.
I was considering around 10µg because in Bionumber database the average RNA yield in mammalian cells is 10 µg (+,- 5µg) but HUVEC are always a little bit different. Thanks a lot to everyone.
Dear Raul,
It will depend a lot of the extraction method. Beware that with guanidinium-based methods (which includes the Trizol) the OD does not necessarily reflect the amount of RNA (in some cases can give you up to 10-fold higher OD). This is due to proteins that are precipitated with the RNA. The way to overcome this is to resuspend the precipitated pellet with 100 µl of a solution of 1%SDS, 50 mM TrisHCl and 10µg/ml proteinase K), incubate for 30 min at 37 and phenol-chloroform-isoamylalcohol extract it, re-precipitating with ethanol.
In general for cell lines we calculate about 1µg/10^6 cells using guanidinium-based methods and about 5g/10^6 cells using NP40 lysis methods (in the latter case you only obtain cytoplasmic RNA, but you preserve small RNAs if you precipitate with 0.3M Na-acetate and 2.5 volumes of etoh)
good luck
- Badges
- Science method