Method for total RNA preparation from Leishmania cells.
(for Beverley lab, August 23, 1999, Natalia S. Akopyants, please contact natalia@wustl.edu for
any questions)
I.
Preparation of Leishmania cells.
Early Log phase cells should be passed once a day for 2 days and then collected @ 1-2 x
10
6
cells/ml for early log, 3-4 x 10
6
cells/ml for mid log and 7-9 x 10
6
cells/ml for late
log.
Metacyclic cells should be collected on 7-8 day of stationary phase.
For amastigotes (from mouse lesions) see method on ‘smb/common/SMB
methods/natalia/method for preparation and purification of amastigotes’.
Wash cell in cold 1 x PBS and dissolve pellet in TRIZOL solution (Gibco-BRL). Here is
an estimation based on my experimental data:
Log phase cells: up to 5 x 10
8
- in 1 ml TRIZOL,
Metacyclic cells: 1-2 x 10
9
- in 1 ml TRIZOL,
Amastigotes cells: 2-3 x 10
9
- in 1 ml TRIZOL
At this point cells could be stored at –80
C for at least 6 months.
Before beginning, make sure the bench and pipets are cleaned with ethanol and then
washed with RNAse Away solution (BRL) or RNA Zap (Ambion). Gather the supplies
needed:
-Isopropanol
-Chloroform
-1 ml syringe
-27G Needle
-RNAse free tubes (1.5 ml and 2.0 ml)
-Cold 75% ethanol
-RT 100 % ethanol
-RNeasy Mini Kit (Qiagen catalog # 74104)
-Dnase Free RNase Kit (Qiagen catalog # 79254)
-RNAse free water
 |