RE: [ High efficiency RNA extraction from low numbers of cells ]

hi

we do this all the time with Trizol and 10,000 cells is sufficient.  So you should pick a
cell line or just use whole BM and work on your technique, including checking the
integrity of the RNA.  The Maniatis guide has good advice for precautions to take....  
You can try doping in tRNA to help protect the mRNA, this often helps

good luck,
Rachel

=======================================================
Rachel M. Gerstein, Ph.D.
Associate Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002
(508) 856-1044
(508) 856-5920 (FAX) 



-----Original Message-----
From: Kapur, Gaurav [mailto:GKapur@dmc.org]
Sent: Tue 1/10/2006 9:44 AM
To: cyto-inbox
Subject: [ High efficiency RNA extraction from low numbers of cells ]
Hi everybody,

I am a new member to the cytometry mailing list and have just started my basic science
research. I am working on sorting SP cells based on Hoechst analysis and would like to
isolate RNA from this small cell population (10,000-20,000cells). I was isolating the
cells directly in Trizol on ice but this is not giving me a good RNA integrity. Does
anybody have a protocol for isolation of small cell populations and then RNA isolation
from them which gives a good yield and quality of RNA. 
I am very new to research and hope somebody can help me with this,
Thanks in advance.

Gaurav Kapur
Children's Hospital of Michigan
Michigan
email: gkapur@dmc.org