Superscript 2 plus rnase h reverse transcriptase

Total RNA was prepared using RNeasy mini kit (Qiagen) and treated with DNase I, Amp Grade (Invitrogen, Carlsbad, CA, USA) prior to cDNA synthesis. RNA integrity was electrophoretically verified by ethidium bromide staining. RNA (1 μg) was reverse transcribed with 100 U of Superscript II plus RNase H⁻ Reverse Transcriptase (Invitrogen) using 100 μM random hexamer primers (Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer's instructions. Real-time quantitative PCR was carried out on a Bio-Rad CFX-96 detection system with quantitative qPCR SYBR Green reagents (Bio-Rad, Hercules, CA, USA) and with a primer concentration of 0.5 μM. For a list of primer sequences, see Supplementary Table 1. PCR conditions were standardized to 39 cycles of: 95°C for 08 s, 59°C for 05 s and 72°C for 10 s.

Total RNA was prepared using a RNeasy mini kit (Qiagen) and treated with DNase I, Amp Grade (InVitrogen, Carlsbad, CA, USA) prior to cDNA synthesis. RNA integrity was electrophoretically verified by ethidium bromide staining. RNA (0.5 µg) was reverse transcribed with 100 U of Superscript II plus RNase H- Reverse Transcriptase (InVitrogen) using 100 µM random hexamer primers (Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer's instructions. Real-time quantitative PCR was carried out on a Bio-Rad CFX-96 detection system with quantitative Q-PCR SYBR Green reagents (Bio-Rad, Hercules, CA, USA) and with a primer concentration of 0.5 µM. PCR conditions were standardized to 40 cycles of 95°C for 10 s and 59°C for 30 s with the primers for specific mouse mRNA sequences (For a list of primer sequences, see Table S1). To control for RNA quality and cDNA synthesis, β-actin or Nono mRNA was also amplified. The abundance of each RNA normalized to the HPRT1 or Nono signal depending on the tissue are expressed as the mean ± SEM of at least 6 samples.

To determine the expression of prophage genes upon biofilm formation, bacterial RNAs were obtained at 24h, days 3 and 5, after M. avium was placed in contact with a polyvinyl chloride plate surface. The Real-Time (RT) PCR was carried out using the conditions as previously described [17 (link)]. Briefly, total bacterial RNAs from broth grown bacteria (control) and from biofilms (experimental) were extracted with the combination of a guanidine thiocyanate-based buffer (Trisol) (Invitrogen, Carlsbad, CA) and rapid mechanical cell lysis of M.avium in a bead-beater. Prior to the real-time PCR, RNA was cleaned up with RNA clean kit (QIAGEN, Valencia, CA) and treated with DNase I. RNA quality was verified by ethidium bromide staining on the agarose gel and by OD_260/280 nm absorption. Mycobacterial total RNA (1 μg) was reverse transcribed with 100U of Superscript II Plus RNase H⁻ Reverse Transcriptase (Invitrogen, Carlsbad, CA), using RT primers according to the manufacturer’s instruction. M. avium gene expressions were quantified with SYBR Green I assay by Real-Time PCR detection system using gene-specific primers.