: What's the best method for isolation of RT-PCR suitable RNA from human serum ?
: Thanks, Christian Schneeberger (FAX +43-1-40400-7832)
This is one method that I read about. I am guessing here that you are trying
to isolate Viral RNA. It's been obsereved many times that heparanized serum
prevents successful RT-PCR. This is especially true of HCV and HIV.
Here is a protocol that I found:
take 50ul of heparanized serum and add 250ul of 3mM Calcium Acetate(PH 7.0)
Add a little bit of Heparinase -> incubate at 37C for 2hrs.
Add 300ul of 6M GITC(in Tris-HCL PH 7.5) and 10ul of Glass fines.
Place at RT for 90min.
Wash Glass fines with 150ul of 20mM Tris-HCL (pH7.4), 1mM EDTA, O.1M NaCl
Wash with 150uL of Cold ETOH; Repeat. This process removes Ca2+ which is
inhibitory to RT.
Elude RNA and PLAY as you like.
C A N A D A
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM