Peqgold rna isolation kit

Liquid nitrogen-frozen tissue was homogenized using a micro-dismembrator (Sartorius, Göttingen, Germany). RNA extraction from mouse tissue was performed using the Roti Quick kit (Carl Roth, Karlsruhe, Germany) followed by RNA purification with the peqGold RNA isolation kit (Peqlab, Erlangen, Germany), according to the manufacturers’ instructions. For RNA quality assessment, RNA integrity numbers (RIN) were measured via Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and samples with RIN number above 6.5 were used for sequencing. RNA library preparation was done using the Illumina TruSeq RNA Sample Prep kit v2 following the manufacturers’ protocol. RNA sequencing was performed as 100-bp paired-end runs on a HiSeq2500 (Illumina). Pools of 16 indexed libraries were sequenced on four lanes. Image analysis and base calling were performed using Illumina Real Time Analysis. CASAVA 1.8 was used for demultiplexing.

For RNA extraction, the peqGold RNA isolation kit (Peqlab, Erlangen, Germany) was used for phLF and the RNeasy Mini Plus Kit (Quiagen; Venlo, Holland) was used for phBEC according to the manufacturer’s instructions. RNA was reverse-transcribed in a 40 μl reaction using M-MLV reverse transcriptase and random hexamers, according to the manufacturer’s protocol (Life Technologies). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green PCR master mix (Roche Applied Science, Mannheim, Germany).
Relative transcript abundance of a gene is expressed as −ΔC_t values (−ΔC_t = C_t^reference − C_t^target) or as fold change derived from the relevant ΔΔC_t values, using 2^−(ΔΔCt). For specific gene amplification, primers listed in Table 2 were used. ATP-dependent RNA helicase DHX8 (DHX8) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) were used as endogenous controls for standardization of relative mRNA expression in phLF and phBEC, respectively. Normalization with both reference genes provided similar results and, for data presentation, analysis was performed using DHX8 as reference gene. In a previous study45 (link) we have assessed the quality of a number of reference genes for lung transcriptomes derived from Genevestigator V368 (link) and found that DHX8 was a reliable reference gene in this context.

Relative abundance of ACE, ACE2, PEP, PCP, renin and NEP mRNAs in murine renal medulla and cortex were analysed by semiquantitative real time PCR. Kidneys were separated into medulla and cortex and further mechanically disrupted in TriFast^®(PeqLab, Erlangen, Germany) using a glass douncer. Total RNA was extracted using the PeqGold RNA isolation kit (PeqLab) and 1 μg of RNA reversely transcribed using QuantiTect^® transcriptases (QIAGEN) and random polyT primers. Resulting cDNA was diluted (1:30) in DNase/RNase free water (Gibco) and directly used in amplification reactions. Predeveloped TaqMan probe/primer pair assays (Supplementary Fig. 4b) and accessory reagents (PCR SuperMix-UDG, ROX reference dye, all from Life Technologies) were used for amplification. PCR reactions were performed at a 10 μl scale on an Applied Biosystems SDS 7900 real time PCR System (Life Technologies) in quadruplicates; relative quantification was performed by the ΔΔCT method (Schmittgen and Livak 2008), normalizing target gene expression on Actb/β-Actin.