RNA extraction from mouse splenocytes in 96 well plate - (Nov/15/2006 )

Anybody have prepared RNA from mouse smc cultured in 96-well plate? I tried twice and found it too little to be detected in Real-time assay.

Here is my protocol: 0.3 million smc plated in 96-well plate. After a scheduled time, RNA is isolated using RNA Easy kit (Qiagen). 30 ul RNase-free water wash down RNA from the column. 10ul rna was reverse-transcripted into cDNA (20ul total). 3 ul cDNA was added into Real Time PCR 96-well plate, 5ul sybergreen(2*), and 2 ul primer (0.1 u mol/L). ABI 7500 system.

Thank you!

did you quantify your RNA?
maybe you loose it or too diluted for a correct exp...