Transcriptor first strand kit

Fat pads were minced in RNA extraction buffer and homogenized in a TissueLyser II (Qiagen, Hilden, Germany). The homogenates were then centrifuged at 12,000 g, for 20 min at 4 °C. The lipid layer was discarded and the aqueous phase collected for RNA purification.
Total RNA was isolated (NucleoSpin® RNA and NucleoSpin® RNA XS, Macherey–Nagel, Düren, Germany) and RNA integrity evaluated (Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA, USA). cDNA was synthetized from RNA samples with RIN > 6 using random primers (Transcriptor First Strand kit, Roche Diagnostics). Semi-quantitative PCR was performed with the LightCycler 480 system (Roche Diagnostics) using specific primers (Invitrogen, Carlsbad, CA, USA) and probes (Roche Diagnostics). All primers and probes are given in Suppl. Table 1. Normalized gene expression was calculated as the ratio of C_t of target transcripts vs Rps13, used as housekeeping gene (2^−ΔCt_, ΔC_t).

RNA extraction from A549 cells grown in 6-well plates was done using High Pure RNA isolation kit (Roche). Transcriptor First strand kit (Roche) was used for cDNA synthesis and qRT-PCR was performed using FastStart Essential DNA Probes Master and gene specific probes from Universal Probe Library (Roche). Lightcycler 96 (Roche) was used for the PCR and quantification of results was done using 2^∧(-ΔΔCT) method. Primer sequences are given in Table 1.Table 1

QRT-PCR primer sequences.

Table 1

Gene	Forward primer	Reverse primer
TRAF2	GAGGCATCCACCTACGATGG	GTCGTTGACTGGCCTCTGAA
KEAP1	TGCCCGGGAGTACATCTACA	AGGGTGAGCTCCTCGAAGAT
BIRC2	ATAGGGTAGCCTGCTTTGCC	CTCCGGTGTTCTGACATAGCA
IL23A	CCACTGGGAGACTCAGCAGA	TTGAAGCGGAGAAGGAGACG
FAS	GGACCCTCCTACCTCTGGTT	TTTGGTGCAAGGGTCACAGT
TRAF1	AAACCTGCTGTTGGGGTTCA	AGCTGGCTCTGGTGGATAGA
TNFΑIP3	TCGACAGAAACATCCAGGCC	TTCGTTTTCAGCGCCACAAG
NQO1	GAAAGGATGGGAGGTGGTGG	TGGCAGCGTAAGTGTAAGCA
GCLM	ACAGCTGTATCAGTGGGCAC	CTCGTGCGCTTGAATGTCAG
HMOX1	GTGCCACCAAGTTCAAGCAG	CAGCTCCTGCAACTCCTCAA
TXNRD1	GCTCAGAGGCTCTATGCAGG	TGGACCCAGTACGTGAAAGC
XIAP	AGTGCCACGCAGTCTACAAA	TTCTGACCAGGCACGATCAC

Firstly, complementary DNA (cDNA) was generated from 1 μg RNA using the Transcriptor First Strand Kit (Roche, Rotkreuz, Switzerland) according to the manufacturer’s protocol. The collected cDNA was stored in aliquots at −20 °C until further analysis. Fluorescence-labeled probe-based multiplex-RT-qPCR was performed using the innuMix qPCR MasterMix Probe (Innuscreen GmbH, Berlin, Germany). The Minor Groove Binder (MGB)-Double Dye TaqMan probes with a black-hole quencher and the primer sequences used for this experiment were designed and synthesized using Eurofins qPCR-ASSAY software, as provided in Table 1. The amplification conditions were: 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min, and 68 °C for 30 s (qTower 3G touch, Analytic Jena, Jena), as provided in Table 2. The expression data were normalized based on the level of expression of the housekeeping gene ribosomal protein L13a (RPL13A). The results were presented as 2^−ΔΔCt as relative gene expression to a fibrin untreated control in a 2D culture without scaffold regarding the investigated cell type. The control group represents the amount 1. The entire experiment was performed with biological triplicates.

RNA was extracted from a pool of six pellets for each condition or treatment. Pellets were snap frozen, QIAzol (Qiagen, Hilden, Germany) added and then homogenized using a PreCellys system homogenizer. Solution was removed and RNA was isolated using QIAzol method according to the manusfacturer’s instructions. Samples were purified and genomic DNA removed via RNeasy Mini kit (QIAGEN) according to manufacturers’ instructions. Total RNA was quantified and 200 ng was reverse-transcribed using Transcriptor first strand kit (Roche, Mannheim, Germany). Quantitative polymerase chain reaction (qPCR) of chondrogenic genes (listed in Table 1) was performed using Real Time PCR Ready Custom designed plates (BioRad Laboratories, Munich, Germany) according to the manufacturer’s instructions [49 (link)]. qPCR reactions were performed using a Biorad CFX96 system (Biorad). Results were analysed using the ΔΔCt method and normalized to the ribosomal protein 13a (RPL13a)—the most stably expressed of the three housekeeping genes (hypoxanthine phosphoribosyltransferase (HPRT), Proteasome subunit beta type-4 (PSMB4) and RPL13a) evaluated [50 (link)]. Gene expression in physioxia was calculated as fold change from that measured under hyperoxia.

Total cellular RNA extracted from NICCs and human islets using a commercial kit was transcribed using random primers (Transcriptor First Strand kit, Roche, Germany). For reverse transcription quantitative real-time PCR (qRT-PCR), normalised gene expression was calculated as ratio of the cycle threshold (C_t) values of target vs housekeeping gene (RPS13) transcripts ( $2^{-\text{∆}{\mathrm{C}}_{\mathrm{t}}}$ ).