Random hexamer primer

RNA samples isolated from cells were quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE) and 700 ng were reverse transcribed using SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA) and random hexamer primers (GE Healthcare, Mississauga, ON). Real‐time PCR was performed using QuantiTect SYBR Green PCR mix (Qiagen) with previously published human β‐actin (100 nmol/L) (Watson et al. 1992) and AQP3 primers (300 nmol/L) (Okahira et al. 2008) synthesized in‐house (University of Calgary DNA Services). The Applied Biosystems 7900HT Fast Real‐time PCR System was used with three programmed steps running in standard mode: heat activation at 95°C for 10 min; 45 cycles of denaturation (95°C), primer annealing (57°C), and extension (72°C); and a dissociation curve. Data were collected using SDS2.3 and analyzed using the ΔΔCt method (Livak and Schmittgen 2001).

RNA was isolated from cells using peqGOLD TriFast (Peqlab) and from tissue cryosections either using Invisorb Spin Tissue RNA Mini Kit (Invitek; for subsequent mRNA analysis) or peqGOLD TriFast (for subsequent miRNA analysis) according to the manufacturers’ recommendations. For mRNA analysis in tissues and cells, reverse transcription of 500 ng RNA into cDNA was carried out using SuperScript II Reverse Transcriptase (Life Technologies) and random hexamer primers (GE Healthcare) according to the manufacturers’ recommendations. For miRNA analysis in tissue samples, a total of 400 ng RNA was reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit and Megaplex RT Primers (Human Pool A; both Life Technologies) which allows for reverse transcription of up to 381 miRNAs in a single reaction.

Total RNA was isolated from sections of fresh-frozen malignant (tumor cell amount ≥70%) and matched non-malignant (tumor cell amount ≤10%) tissue samples using the Invisorb Spin Tissue RNA Mini Kit (Stratec Molecular, Berlin, Germany) according to the manufacturer's instructions. RNA quality and quantity was determined with the Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany). Up to 500 ng total RNA were reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (200 U; Life Technologies, Darmstadt, Germany), dNTP mix (10 pmol of each dNTP; GE Healthcare, Freiburg, Germany) and random hexamer primers (200 ng, GE Healthcare) according to manufacturers' recommendations.

Extraction of total RNA from quick-frozen biopsies were performed using the NucleoSpin total RNA/Protein isolation kit (Machery-Nagel, Düren, Germany) according to the manufacturer’s instructions. Total RNA was eluted in a volume of 60 μl H₂O. Genomic DNA was digested for 15 min with 1.5 U of DNAse I (Sigma-Aldrich, Munich, Germany) at room temperature. Reverse transcription was carried out in a total volume of 30 μl containing 375 ng random hexamer primers (GE Healthcare, Freiburg, Germany), 0.5 mM dNTPs (Promega, Mannheim, Germany), 0.01 M DTT, 1 x reaction buffer, and 150 U Superscript II Reverse Transcriptase (Invitrogen, Karlsruhe, Germany). The annealing step was carried out at 25 °C for 10 min, elongation at 25 °C for 10 min, and denaturation at 70 °C for 15 min.

Total RNA of 2–4 dpf zebrafish embryos was extracted using Trizol reagent and purified using the RNeasy Mini Kit (Qiagen), according to manufacturer’s instructions. cDNA synthesis was performed with 500 ng RNA in the presence of first strand buffer, 0.75 μg oligo-dT, 0.75 μg random hexamer-primer, 50 nmol dNTPs (GE Healthcare Life Sciences), 1 U RNAse inhibitor (RNAsin, Promega) and 5 U reverse transcriptase for 60′ at 42°C, followed by 5′ at 95°C. The effect on tfam splicing was assessed using RT-PCR amplification of 25 ng cDNA in a PCR mix contained under standard conditions, using 0.6 μM forward primer, 0.6 μM reverse primer (Supplementary Table S1). PCR conditions were 5′ denaturation at 94°C, 35 cycles of 1′ at 94°C, 1′ at 58°C and 1,5′ at 72°C, followed by 7′ at 72°C. The PCR product was sequenced by conventional Sanger sequencing. tfam gene expression was quantified by comparing the RNA expression ratio of Tfam RNA to 18S RNA. Per reaction, 2.5 ng cDNA was amplified in a 10 μl reaction containing 1× Sensimix Sybr Hi-Rox reagents (Bioline) and 375 nM of both forward and reverse primer (Supplementary Table S1). Real-time PCR was performed on an ABI7900HT using the following settings: 10′ 95°C, 40 cycles of 15” 95°C and 1′ 60°C. The statistical analysis was performed by a Student’s t-test. p-values < 0.05 were considered significant.

Extraction of total RNA from human myenteric ganglia (MP), circular (CM) and longitudinal muscle (LM) was performed under usage of the peqGOLD MicroSpin Total RNA Kit (PEQLAB, Erlangen, Germany). Myenteric nerve cell cultures RNA was isolated by the Nucleospin XS kit (Macherey and Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA was eluted in a volume of 15 μl H₂O. Prior to reverse transcription (RT), contaminating genomic DNA was digested for 15 min at room temperature using 1.5 U of DNase I (Sigma-Aldrich, Munich, Germany). RT was carried out in a total volume of 30 μl containing 375 ng random hexamer primer (GE Healthcare, Freiburg, Germany), 0.5 mM dNTPs (Promega, Mannheim, Germany), 0.01 M DTT, 1 × reaction buffer, and 150 U Superscript II Reverse Transcriptase (Invitrogen, Karlsruhe, Germany). Annealing, elongation, and denaturation were carried out at 25°C for 10 min, 42°C for 50 min and at 70°C for 15 min, respectively.