Description and Procedure Overview
This protocol uses TRI Reagent and silica filters for isolation of total RNA from leukocytes captured on LeukoLOCK™ leukocyte depletion filters (Ambion Cat #1933). Adjusting the amount of ethanol used to bind the RNA to the silica filter, determines whether total RNA that includes the small RNA fraction, or RNA that is depleted of smaller species such as microRNA (miRNA), tRNA, and 5S/5.8SrRNA is recovered. The quality and purity of the RNA recovered using this protocol is similar to that seen using the LeukoLOCK Total RNA Isolation System (Cat #1923), and the yields are generally higher by ~50%–100%. Microarray experiments have not been carried out using RNA extracted with this protocol, but it is not expected that alterations in the RNA profiles will occur provided the captured leukocytes are treated with RNA later immediately after filtration of the blood.
Prepare Wash 1
Prepare Wash 1 according to the table below (enough for ~65 preps). Store in a tightly closed container at room temperature.
| Composition || for 50 ml || Component |
|30%||15 ml||Denaturing Lysis Solution (Ambion Cat #8540G)|
|70%||35 ml||100% ethanol|
Prepare Wash 2/3
Prepare Wash 2/3 according to the table below (enough for ~65 preps). Store in a tightly closed container at
Preheat nuclease-free 0.1 mM EDTA to 80°C
| Composition || for 100 ml || Component |
|80%||80 ml||100% ethanol |
|19 ml||nuclease-free water|
|50 mM ||1 ml ||5 M NaCl |
| || || |
Heat an aliquot of nuclease-free 0.1 mM EDTA in a nuclease-free tube to 80°C in a heat block; it will be used to elute RNA from the Spin Cartridges in step E.10 at the end of the procedure. Samples are typically eluted in 250 μl. Do not heat in non-stick tubes.