Fastquant reverse transcriptase kit

Genomic DNA was isolated from ear tissue using the standard phenol/chloroform extraction method, dissolved in a TE solution, and stored at −20 °C. Total RNA was extracted from tissues and cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and its quality and integrity were verified using a NanoDrop 2000 Biophotometer (Thermo Fisher Scientific Inc, West Palm Beach, FL,USA) and via electrophoresis. RNA samples (2 μg) in a 20 μL reaction volume were reverse transcribed to cDNA using the FastQuant Reverse Transcriptase Kit (TIANGEN, Beijing, China).

Complementary DNA (cDNA) was generated from total RNA (1 μg) extracted from P. aeruginosa PAO1 cells with the FastQuant reverse transcriptase Kit (TianGen, China) according to the manufacturer’s instructions. This cDNA was then used as a template for various PCRs. Sequence data obtained from the DOE Joint Genome Institute (JGI) website (www.jgi.doe.gov) were used to design qRT-PCR primers. All qRT-PCR primers (See Supplementary Table S1) were designed according to the manufacturer’s specifications (amplicon sizes of 100 to 200 bp), and representative products from each of these primer sets were verified by sequencing. qRT-PCR amplification and detection were performed with the 7500 real-time PCR system (Applied Biosystems). Optimal qRT-PCR conditions were determined using the manufacturer’s guidelines. Each PCR mixture consisted of a total volume of 25 µL and contained 1.5 µL of the appropriate primers (stock concentrations, 15 µM) and 12.5 µL SYBR green PCR master mix (Bio-Rad).