Stepone plus 96 well rt pcr system

Quantitative RT–PCR (qRT–PCR) was performed as we previously described (61 (link)). RNA for real-time qRT–PCR was prepared using an Eastep Super Kit following manufacturer’s protocols (Promega Co) and treated with DNase I according to the manufacturer’s instructions (Promega Co) prior to complementary DNA preparation. Complementary DNA was prepared using GoScript Reverse Transcription System as described by the manufacturer (Promega Co). qRT–PCR was performed in a 20 μl reaction volume using an Applied Biosystems StepOne Plus 96-well RT–PCR system with Power SYBR Green PCR Master Mix following the manufacturer’s instructions (Applied Biosystems Co). 16S rRNA was used as the reference sample in all comparative threshold cycle (ΔΔCT) experiments. All qRT–PCR primers were tested for amplification efficiency. qRT–PCR data were analyzed using StepOne System software (Applied Biosystems, Inc) and GraphPad Prism (GraphPad Software, Inc). Primers used in qRT–PCR experiments are shown in supplemental Table S19. All analyses were performed in biological triplicate.

Total RNA from lung tissue homogenate was isolated using the peqlab-Gold Total RNA-Kit (Peqlab, Erlangen, Germany, #12-6834-01) before cDNA synthesis (random hexamers, MuLV reverse transcriptase (Applied Biosystems, Germany)). mRNA expression of target and housekeeping gene (hypoxanthine-guanine phosphoribosyltransferase, HPRT-1) expression was determined by Platinum SYBR Green qPCR SuperMix (Applied Biosystems; StepOnePlus 96 well RTPCR System (Applied Biosystems, CA) and displayed as 2^−ΔCT (ΔC_T = C_{T target}–C_{T reference}) or as 2^−ΔΔCT values (ΔΔC_T = ΔC_{T treated}–ΔC_{T control}).