Supplemental Digital Content 1 - Postmortem analysis methods
Molecular biology methods: RNA analysis
Extraction of the total RNA
Total RNA was isolated from the cryopreserved samples by phenol/chloroform extraction.
The lung tissue samples (50-100 mg) were smashed and mixed with 200 μl of Tri-Reagent
(Sigma). Thereafter 800 μl of the Tri-Reagent was added and the mixture was centrifuged
(12.000 x g) for 10 minutes at 4°C. The clear supernatant was transferred into a new 2 ml
Eppendorf tube, 500 μl of chloroform were added and mixed. The suspension was incubated
at room temperature for 10 minutes followed by a centrifugation step (12.000 x g, 4°C, 5
minutes) to separate the phases. The top aqueous RNA phase was removed and transferred to
a new 1.5 ml Eppendorf tube. 500 μl of isopropanol was added and incubation was performed
for 10 minutes at room temperature, followed by 5 minutes of centrifugation at 12.000 x g
and 4°C for the precipitation of the RNA. The supernatant was discarded, the RNA pellet was
washed with 70% ethanol (in DEPC-H2O), and air dried. Lastly, the pellet was dissolved in
40 μl of DEPC-H2O and the extracted RNA stored at -80°C.
DNase treatment and purification of DNA
The extracted RNA was treated with DNase to reduce the contamination with genomic DNA,
and a subsequent purification with NucleoSpin ® RNA II Kit (Macherey & Nagel, Düren,
Germany) was performed according with the procedures described in the enclosed manual
(January 2010/Rev. 11).
Standard polymerase chain reaction (PCR)
The PCR of total RNA was performed to evaluate the effectiveness of DNase treatment. The
PCR was conducted using primers for the gene β2-microglobulin (β2-MG), a 5 Prime Master
Mix (5 Prime, Hamburg, Germany). Details of the procedures are described in Tables SDC1.1, SDC-1.2, and SDC-1.3.
Table SDC-1.1: List of oligonucleotides
Oligonucletides
5' - 3' Sequence
p-Cyclo-A s
p-Cyclo-A as
p-β2-MG s
p-β2-MG as
p-TNF-α s
p-TNF-α as
p-IL-1β s
p-IL-1β as
p-IL-6 s
p-IL-6 as
p-IL-8 s
p-IL-8 as
p-TNC s
p-TNC as
p-AREG s
p-AREG as
Oligo(dt) 15
CTT TCA CAG AAT AAT TCC AGG ATT
GGA CAA GAT GCC AGG ACC
AAG GTT CAG GTT TAC TCA CGC CAC
GCG GAT GGA ACC CAG ATA CAT AGC A
CGT TGT AGC CAA TGT CAA AGC CGA
TGT CTT TCA GCT TCA CGC CGT T
TGA AAG ATA ACA CGC CCA CCC T
TGT TCT GCT TGA GAG GTG CTG A
TGT CGA GGC TGT GCA GAT TAG T
ACT CGT TCT GTG ACT GCA GCT T
CCT TCT TGG CAG TTT TCC T
GTG GAA AGG TGT GAA ATG C
TGC TCC CAA GCA ATG CTG AAT GGA
TTC CAG CTT CTG GGC TTT GTC GTT
ATT ATG CTG CTG GAC TGG AC
TCG CTA CCA GAA GGC ATT T
TTT TTT TTT TTT TTT
p = pig; s=sense, as = antisense, Cyclo-A=cyclophilin-A, β2-MG= β2-microglobulin, TNF-α=tumor necrosis
factor – alpha, IL-1β=interleukine - 1β, IL-6=interleukine – 6, IL-8=interleukine – 8, TNC=tenascin C,
AREG=amphiregulin.
Table SDC-1.2: Reaction conditions
Reaction conditions:
1 μl total RNA
1 μl Sense primer
1 μl Anti-sense primer
10 μl 5 Prime Mastermix
(2.5x)
12 μl DEPC-H2O
Table SDC-1.3: Standard PCR Program
Reaction steps
Initial denaturation
Denaturation
Annealing
Elongation
Syntheses
Time
5 min
30 sec
30 sec
30 sec
5 min
Temperature
95°C
95°C
56°C
72°C
72°C
Number of cycles
1
30
1
Agarose gel electrophoresis of DNA
The agarose gel electrophoresis was performed to verify the presence of the PCR fragments
and separate them. The concentration of agarose was based on the size of the fragments,
which was determined applying a 50 bp ladder. The DNA samples were prepared with 1 x
sample buffer. The gel run was carried out with 7-10 volts per cm electrode gap. After
completion, the agarose gel was photographed under UV light with a wavelength of 312 nm.
Details of the components used for this procedure are described in Table SDC-1.4.
Table SDC-1.4: Components of the agarose gel, 50 x TAE, and sample buffer.
Agarose gel
50 x TAE
(pH 8.0)
6 x Sample buffer
1% to 2% (w/v) agarose
50 mM EDTA
0.2% (w/v) Bromphenolblue,
Sodium-salt
1 x TAE used and mixed
with Gel Red
1 M acetic acid
0,1 M EDTA
33% (v/v) Glycerin
The cDNA synthesis
For cDNA synthesis, a reverse transcription was performed from the total RNA. The amount
of 2 μg from the total RNA was transcribed in cDNA using Revert AidTM H Minus First
Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon Roth, Germany). The cDNA was
stored at -20°C. An overview of the reaction mixture is described at
Table .
Quantitative Real Time PCR
The quantitave determination of mRNA expression was performed using a MyiQ2 ® (Bio Rad, Munich,
Germany). The reaction mixture and quantitative real time PCR program are described at Table and
Table -1.7, respectively.
Table SDC-1.5: cDNA sythesis.
Reaction steps
A
B
C
D
Reagents
Volume
RNA (for 2 μg)
Oligo(dT)12-18
x μl
2 μl
y μl
24 μl
On ice
5x Reaction buffer
8 μl
dNTP’s
4 μl
RNase-Inhibitor
2 μl
Reaction volume
38 μl
Reverse Transcriptase
2 μl
Reaction volume
40 μl
10 mM Tris/HCl
40 μl
pH = 7,4
Final volume
80 μl
DEPC-H2O
Reaction volume
Duration
Temperature
5 min
70°C
5 min
37°C
60 min
10 min
42°C
70°C
-
-
Table E-1.6: Reaction Mixture
Reaction conditions:
7,5 μl Mastermix 2x
1,5 μl cDNA
0,15 μl FITC (1 μM)
4,95 μl ddH2O
0,9 μl Primer sense und antisense (per 5 pmol/μl)
15 μl
Final volume
Table SDC-1.7: Quantitative real time PCR Program (qRT-PCR)
Reaction step
Duration
Temperature
Number of cycles
Initial denaturation
Denaturation
Annealing
Elongation
Synthesis
2 min
15 sec
20 sec
15 sec
2 min
95 °C
95 °C
58 °C
72 °C
72 °C
1
45
1
Biochemical methods: Protein Analysis
Protein extraction
The cryo-preserved tissue was pulverized at -80°C and protective buffer (4°C) was added
(components of the protective buffer are shown in Table SDC-1.8). Subsequently, the
homogenization (750x, 1 min) and centrifugation (5000 x g, 5 min) at 4°C were performed.
The supernatant was aliquoted and stored -80°C.
Table SDC-1.8: Components of the protective buffer
20 mM
Bis/Tris
5 mM
DTT
90 mM
NaCl
10%
Glycerin
1 Protease inhibitor tablet per 10ml buffer.
pH 6,7
Diffuse Alveolar Damage (DAD) Score
Pictures were taken at 25x, 100x and 400x magnification from four non-overlapping fields of
view, which were selected to avoid bronchi and vessels. The images were examined using a
microscope (Leica DMRB, Wetzlar, Germany) and Sony DXC-950P camera (Sony, Tokyo,
Japan). For processing and recording of digital images, the program AnalySIS™ Version 3.2
was used.
Diffuse alveolar damage scoring (DAD score) was performed by one of the authors (MK),
who is an expert in lung and was blinded to the group assignment, using a weighted scoring
system similar to that proposed in a previous study of our group (1). Values from 0 to 5 were
used to represent the severity of the features alveolar edema, interstitial edema, hemorrhage,
alveolar infiltration, interstitial infiltration, epithelial destruction and overdistension, with 0
standing for no effect and 5 for maximum severity. In addition, the extent of involvement in
each field of view was determined with values of 0 to 5, with 0 standing for no involvement
and 5 for complete involvement. The contribution of each feature was calculated as the
product of severity and involvement, being situated in the range 0-25, while the cumulated
DAD score was calculated as the sum of all features (range 0-175).
Reference
(1) Gama de Abreu M, Quelhas AD, Spieth P et al: Comparative effects of vaporized
perfluorohexane and partial liquid ventilation in oleic acid-induced lung injury.
Anesthesiology 2006; 104: 278-89