Supplemental Protocols
Protocol 1: RNA isolation
Blood collection
TM
Blood was drawn in PAXgene Blood RNA Tubes, that contain a proprietary reagent
composition based on a patented RNA stabilization technology that time stabilizes
intracellular RNA. By that, ex vivo changes of expression profiles are avoided. The
RNA can be stabilized by PAXgene™ system for three days at 18-25°C, for five days
at 2-8°C, and over a longer period (at least 6 months) at -20°C to -70°C. The
PAXgene™ tubes contain 6,9 ml of RNA stabilizing solution and are suitable for the
sampling of 2,5 ml blood per tube.
2,5 ml to 5 ml blood were sampled from each patient and healthy blood donor. After
blood withdrawal either RNA isolation was performed directly or tubes were stored
over night at 4°C. For a longer storage over several days tubes were frozen at -20°C.
Before RNA isolation the blood was incubated for at least 2 hours at room
temperature (RT).
Isolation of total RNA from blood cells
Blood cells were pelleted by centrifugation (5000g, 10 min, RT). The pellet was
resuspended in 10 ml RNase free water, and centrifuged again. The total RNA
including the miRNA was isolated and purified using the miRNeasy Mini Kit (Qiagen).
The pellet was resuspended in 700 µl QIAzol lysis reagent and incubated for 5 min at
RT. Then 140 µl chloroform were added, vortexed for 15 sec, and incubated for 2-3
min at RT. After centrifugation at 14000 rpm and 4°C for 15 min. the upper, watery
phase was removed and mixed with 1,5 vol 100% ethanol to precipitate the RNA.
Each 700 µl of this mixture were placed on a column and centrifuged at 13000 rpm at
RT for 15 sec. After the mixture had completely passed the column, 700 µl of Buffer
RWT were added, and centrifuged at 13000 rpm at RT for 15 sec. 500 µl Buffer RPE
were added to the column and centrifuged at 13000 rpm at RT for 15 sec. After this,
another 500 µl Buffer RPE were added to the column and centrifuged at 13000 rpm
at RT for 2 min. The column was then dried by centrifugation at 13000 rpm and RT
for 1 min. RNA was eluted with 40 µl RNase free water. The RNA was stored at 70°C until use.
Protocol 2: Microarray analysis
300 ng of total RNA was mixed and 1 µl of 5 pM miRNA spike-in mix and dried in a
table top speedvac. Each RNA pellet was fully resuspended in 25 µl of this
hybridization buffer and denatured for 3 minutes at 95 °C. Until the hybridization, the
denatured samples were kept on ice.
Microarray hybridization was performed using the Geniom® RT Analyzer and
Geniom® miRNA biochips homo sapiens. Samples were loaded automatically and
the hybridization (14 hours, 42°C) was started. After hybridization, SAPE solution,
antibody solution, equilibration buffer (1xNEB2, New England Biolabs), stop buffer
(6xSSPE) and enzyme solution were place into the RT Analyzer.
The array equilibration was followed by incubation with enzyme solution. Enzyme
incubation was stopped with stop buffer. SAPE staining, signal amplification and
detection proceeded fully automated within the Geniom® RT Analyzer.
Spike-in mix:
107:5’-GCAAAGGCUAUCGUCAAGAGAUC-3’
142:5’-GUCGGCAUUUGGCUGGAACUUCAUA-3’
170:5’-UGACGGGUCUCUUCUUCGAUAGC-3’
30:5’-CAAAUCAACAAGAUGAGGUCUGGGG-3’
327:5’-CUUCCUGACCUUACCGAUUCCGA-3’
35:5’-UCAUUGCCUACAAGCCACCAAGC-3’
41:5’-GACAAAUCGGAUUCAAGGGCAGG-3’
46:5’-AGAUGUGGUUGCAACUUCGGAGC-3’
473:5’-UACCAACCCCACCAAAACCAAACGU-3’
509:5’-UCCAAAACCAAACCAAAUCCAAACC-3’
576:5’-ACAACCACUACUUCCGCCGUCAA-3’
610:5’-AACUCAAGCCGCCGGAAUCUUCA-3’
63:5’-AACACCCGUCAAGUCCAGUGCAU-3’
75:5’-UGCGCGGACUCCAACACUUUGUU-3’,
92:5’-UGAUUGUUGUGACACCGGCACUACU-3’
hybridization buffer for eight arrays:
66µl 20x SSPE
22 µl 100% Formamide
22 µl 1x TE buffer
4.4 µl BSA 50 mg/ml
4.4 µl 0.5 % Tween-20
5.5 µl shortmer control oligo mix 1:30
95.7 µl DEPC water
shortmer control oligo mix:
F2.1-Cy3:5’-[Cy3]TCACTCATGGTTATGGCAGCACTGC-3’ 80 nM
F2.2-Bio 5-[bio]GTAGTTCGCCAGTTAATAGTTTGCG-3’ 12 nM
F2.3-Bio 5’-[bio]TCTTACCGCTGTTGAGATCCAGTTC-3’ 4 nM
F2.4-Bio 5’-[bio]CCCACTCGTGCACCCAACTGATCTT-3’ 0.4 nM
F2.5-Bio 5’-[bio]CCATCCAGTCTATTAATTGTTGCCG-3’ 0.04 nM.
SAPE solution:
9 ml 6x SSPE
44 µl SAPE
1 mg/ml; 360 µl BSA 50 mg/ml
antibody solution:
1750 µl 2x stain buffer (41.7 ml 12x MES; 92.5 ml 5M NaCl; 25 ml Tween-20; 90.8 ml
DEPC water)
140 µl BSA 50 mg/ml
35 µl Goat IgG
21 µl biotinylated Anti-streptavidin antibody
1554 µl DEPC water
enzyme solution:
44 µl NEB2, 10x
44 µl Bio-dATP, 40 µM
2.9 µl Klenow exo-, 50000 U/ml
349.1 µl DEPC water