RNA extraction from small cell number - (Apr/27/2006 )

Hi,
I have to extract RNA from a sample with 10.000 cells to perform cDNA and Realtime-PCR. I have to extract, at least, 1ug of RNA to make Retrotranscripasse (I think that I can extract less than 1 ug). Which kit do you recomend me? I would like a kit that include DNAse treatment before elute RNA. Are there some trick to make retrotranscriptasse more efficience?

Thanks

-sus12-

The kit I use (Fermentas) says we can reverse transcribe from as less as 100ng of total RNA. If you browse through different company kits for RNA isolation some go as low to even pico levels.

-Calvin*-

i would recommend trizol with 500µl trizol.
After adding the choloroform and spin, pipett the supernatant and add 1µl of an 100mg/ml glycogen solution, mix and isopropanol. pellet then and wash with etoh 70 (1ml recommended, for getting out most of the glycogen).
Glycogen remaining does not interfers with qRTPCR or OD readings.

-fred_33-

QUOTE (fred_33 @ Apr 28 2006, 09:50 AM)

add 1µl of an 100mg/ml glycogen solution,

Just curious....Is the glycogen to aid in precipitation of the RNA? Would adding glycogen at that stage when doing a trizol extraction from a larger amount of cells also help, or is that ony useful when using small number of cells?

-poxvirus-

hi
glycogen is a carrier used for RNA precipitation of low quantities. I don't use it when cell number is ok.

-fred_33-