Female rats [Wistar, Crl:WI (Wu), nulliparous and non-pregnant] and Balb/c mice [nulliparous and non-pregnant] were housed under conventional and certified laboratory conditions in a regular 12-h dark/light cycle at an ambient temperature of 22 ± 2 °C and a relative air humidity of 55 ± 15%. Diet and drinking water were available ad libitum. Animals were acclimated for at least 1 week and sacrificed by an i.p. overdose (~100 mg/kg body weight) of pentobarbital sodium (Narcoren®, Merial GmbH, Hallbergmoos, Germany) at the age of 10–12 weeks.
Lungs of common marmosets (Callithrix jacchus) and rhesus macaques (Macaca mulatta) were obtained from German Primate Center (Göttingen, Germany). Marmosets were anesthetized using diazepam (Ratiopharm, Ulm, Germany) and alfaxalone (Alphaxan®, Jurox (UK) Limited, Worcestershire, United Kindom) followed by a lethal dose of pentobarbital sodium (Narcoren®, Merial GmbH, Hallbergmoos, Germany) under deep general anesthesia. Rhesus macaques (Macaca mulatta) were deeply anesthetized using a combination of ketamine (Ketavet®, Pfizer, New York, USA) and xylazine (Rompun®, Bayer, Leverkusen, Germany). The abdominal cavity was opened along the linea alba, and the abdominal aorta was cannulated for blood sampling. The aortic cannula was subsequently used to administer a lethal dose of pentobarbital sodium (Narcoren®, Merial GmbH, Hallbergmoos, Germany).
Human lung tissue
Human lung lobes were obtained from male and female patients who underwent lung resection for cancer. Tumor free tissue was processed immediately on the day of resection as described below. The average age of patients was 60 ± 10 years, and 80% of them were smokers.
PCLS preparation, cultivation, and storage
Mouse lungs were filled in situ. Rat, human, and non-human primate lungs were filled ex situ and PCLS were prepared as previously described [5, 21, 22]. Briefly, the trachea was cannulated and the lungs were filled up with 37 °C-warm, 1.5% low-gelling agarose medium solution (Sigma-Aldrich, Munich, Germany). After polymerization of agarose to gel, lung lobes were cut into 200- to 300-µm-thick slices using a Krumdieck microtome (Alabama Research and Development, Munford, AL, USA) filled with 4 °C-cold EBSS (Sigma-Aldrich, Munich, Germany). Subsequently, precision-cut lung slices were incubated in DMEM (Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham (DMEM, pH 7.2-7.4) with l-glutamine and 15 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) without phenol red and fetal bovine serum supplied from Gibco™ (Life Technologies/Thermo Fisher Scientific, Dreieich, Germany) supplemented with 100 units/mL penicillin and streptomycin (Lonza, Verviers, Belgium) under standard cell culture conditions (37 °C, 5% CO2, 100% humidity). Two PCLS were cultured together in 500 µL DMEM as described previously . Different numbers of slices were pooled, as outlined in the RNA isolation section below, immediately transferred into liquid nitrogen and subsequently stored at −80 °C.
Isolation of cells from PCLS
After preparation each PCLS was placed in 200 µL digestion solution (DMEM supplemented with 1% penicillin and streptomycin, 100 U/mL DNase I (Roche Diagnostics, Mannheim, Germany), 2.4 U/mL Dispase® II (Roche Diagnostics, Mannheim, Germany), and 150 U/mL collagenase 3 (Collagenase Worthington, Lakewood, USA)) and incubated for 30 min at 37 °C on an orbital shaker (650 rpm). Afterwards, slices were passed vigorously through a cut 1-mL tip and placed in a 100-µm CellTric® (Partec, Görlitz, Germany). Cells were rinsed with 600 µL ice-cold DMEM per slice, centrifuged for 3 min at 400×g at 4 °C, then immediately transferred into liquid nitrogen, and subsequently stored at −80 °C.
The human lung epithelial cell line A549 (ATCC® CCL-185™) was obtained from ATCC (LGC Standards GmbH, Wesel, Germany). Cells were routinely cultured in 75-cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies/Thermo Fisher Scientific, Dreieich, Germany) supplemented with 10% fetal calf serum and 0.01% gentamicin at 37 °C in a humidified atmosphere containing 5% CO2. Cell numbers used for RNA isolation are indicated in Figs. 2a and 3b. RNA from A549 cells was used in some experiments as a quality standard for comparison.
RNA isolation protocols
In a first step, four rat PCLS per tube were used for RNA isolation. Several commercially available kits were used for this according to the suppliers’ instructions: RNeasy Mini Kit (Qiagen, Hilden, Germany), QIAzol® (Qiagen, Hilden, Germany), MagJET (Thermo Fisher Scientific, Dreieich, Germany), and MagMAX™ (Ambion™/Thermo Fisher Scientific, Dreieich, Germany). Disruption and homogenization of PCLS in the respective solutions was performed using an Ultra-Turrax® (T8, IKA, Stauffen, Germany).
In a second step, optimized RNA extraction was achieved as follows: two PCLS were pooled, followed by disruption and homogenization of PCLS in 400 µL RLT lysis buffer (Qiagen, Hilden, Germany) using an Ultra-Turrax®. The homogenate was transferred to 1 volume of phenol/chloroform, carefully shaken for 30 s, and centrifuged for 5 min at 12,000×g. Subsequently, 1 volume of chloroform/isoamyl alcohol was added, again carefully shaken for 30 s, and centrifuged for 5 min at 12,000×g. The aqueous phase was transferred and RNA was cleaned up with MagMAX™ magnetic beads including the spin procedure step according to the supplier’s instructions. Total RNA was dissolved in RNase-free water and stored at −80 °C. For some samples an additional clean up step was performed using the RNeasy Mini Kit clean up protocol (Qiagen, Hilden, Germany).
RNA from A459 cells was isolated with the commercially available RNeasy Mini Kit according to the supplier’s instructions.
RNA measurements, quality control and quality criteria
RNA concentration (A260) and purity (A260/A280 ratio) were measured by spectrophotometry (NanoDrop 1000 Spectrophotometer, version 3.7, Thermo Fisher Scientific, Dreieich, Germany). RNA integrity (RIN) was evaluated using an Agilent 2100 Bioanalyzer® (Agilent Technologies, Ratingen, Germany).
Quantitative real time RT-PCR analysis (RTqPCR)
Reverse transcription (RT) of RNA was performed using TATAA GrandScript cDNA Supermix (A103a/A103b, TATAA Biocenter, Gothenburg, Sweden). SYBR Green (TATAA SYBRGrandMaster Mix ROX (TA01-1875R, TATAA Biocenter, Gothenburg, Sweden) was used as a fluorescent dye to determine the amplified PCR products after each cycle. The following primer pairs were used: B2M (NM_004048.2), fwd: GAGGCTATCCAGCGTACTCCA, rev: CGGCAGGCATACTCATCTTTT, 248 bp, (Invitrogen/Thermo Fisher Scientific, Dreieich, Germany); MUC5AC, #qHsaCID0017663, 144 bp (BioRad, Munich, Germany). qPCR conditions were as follows: 30 s 95 °C; 5 s 95 °C/30 s 60 °C, for 40 cycles; and 15 s 95 °C/1 min 60 °C/30 s 95 °C for the melting curve. At the end of each extension phase, fluorescence was recorded and at the end of a run quantification cycles (Cq) were determined for each sample. Serial dilutions of RT reactions (A549 for B2M, human PCLS for MUC5AC) were prepared in triplicate and samples were analyzed by qPCR to measure the Cq values. A plot of Cq values versus the logarithm of target concentrations resulted in standard curves, which were used for efficiency calculations (10−(1/slope) − 1, corresponding to 100%) [23–25]. To calculate the efficiency for several individual RNA samples, RNA was transcribed to cDNA (complementary DNA), two dilution steps within the log-linear portion of the standard curve separated by factor 10 were prepared (1:5, 1:50), and Cq values were measured. The Cq difference represents the slope, which was then used for efficiency calculation.
Microarray analysis was performed with Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays, using 250 ng RNA as input. All steps were performed according to the manufacturer’s instructions for the GeneChip® platform (3′IVT PLUS Reagent Kit, Affymetrix, Santa Clara, USA). The steps included first-strand cDNA synthesis, second-strand cDNA synthesis, synthesis of labeled cRNA by in vitro transcription, purification of labeled cRNA, fragmentation, array hybridization, washing, staining, and final scanning of the arrays using the GeneChip® Scanner 3000 7G.
To examine the quality of the microarrays before and after normalization, we used the bioconductor package arrayQualityMetrics 3.24.0  under R version 3.2.1 together with R Studio . For quality assessment two different automatically created HTML reports were interpreted. The results included between-array comparisons, array intensity distributions, variance mean dependence, and finally the individual array quality. Outlier detection considering the distances between array comparisons was performed by finding arrays for which the sum of the distances from all other arrays was exceptionally large. Outlier detection for the array intensity distributions was performed by computing the Kolmogorov–Smirnov statistic Ka between distributions of intensity values of all samples in each array and the distribution of the values in the pooled data. The threshold was determined to be 0.0249. Regarding the individual array intensity, the mass of the distribution in an MA plot is expected to be concentrated along the M = 0 axis with no trend in M (log ratios) as a function of A (mean average). Outliers were detected by computing Hoeffding’s statistic Da on the joint distribution of A and M for each array. The normalized data were imported into the geneXplain platform (www.genexplain-platform.com) to perform a principal component analysis (PCA). The geneXplain platform was further used to detect differentially expressed genes for all samples with the EBarrays workflow.