Supplement 2
Detailed description of RNA isolation procedures
RNA Isolation with TRIzol®
1. After thawing, 0.2 ml of chloroform per ml of the TRIzol® Reagent was added to each
sample. The tubes were shaken by hand for 15 seconds and incubated for 3 minutes
at room temperature. The samples were centrifuged at 13,000 rpm in a
microcentrifuge for 10 minutes at 4°C.
2. The upper aqueous phase was transferred to a fresh tube and the RNA was
precipitated by the addition of 0.5 ml of isopropanol per ml of the TRIzol® Reagent
used in the initial homogenization. The samples were incubated for 10 minutes at
room temperature and centrifuged (13,000 rpm, 30 min, 4°C).
3. The supernatant was removed and the RNA pellets were washed twice by adding 1
ml of 80% ethanol, mixed by vortexing and centrifuged (13,000 rpm, 5 minutes, 4°C).
4. After washing, the RNA pellets were air-dried for 5-10 minutes, dissolved in RNAasefree water and incubated for 5 -10 minutes at 55°C. The RNA was then purified with
the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany).
RNA isolation with PAXgeneTM Blood RNA Isolation System
7.5 ml of peripheral blood were collected by venipuncture using three PAXgene Blood RNA
Tubes (Qiagen, Hilden, Germany). Total RNA was prepared following the manufacturer’s
recommended procedures, including an optional DNase digestion step (RNase-Free DNase
Set, Qiagen). The isolated RNAs were pooled and then concentrated with the RNeasy
MinElute Cleanup Kit (Qiagen).
RNA isolation with QIAamp® RNA Blood Mini Kit
9-18 ml of peripheral whole blood were collected by venipuncture in sodium citrate tubes
(Sarstedt, Germany) and further processed within 15 min. Blood samples were divided into
aliquots with up to 1.5 ml volume containing maximal 1 x 107 cells and were processed with
the QIAamp RNA Blood Mini Kits (Qiagen, Hilden, Germany) following the manufacturer’s
recommended procedures, including an optional DNase digestion step (RNase-Free DNase
Set, Qiagen). The isolated RNAs were pooled and then concentrated with the RNeasy
MinElute Cleanup Kit (Qiagen).