Transcriptor first strand cdna synthesis kit for rt pcr

1 μg of total RNA, extracted with TRI Reagent (Ambion, Life Technologies), was reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit for RT-PCR following the indications of the manufacturer (Roche). Real-time PCR was conducted with SYBR Green (Roche) on a MyiQ Real-Time PCR System (Bio-Rad). The TaqMan probes for mouse EP1, EP2, EP3, EP4, P2Y2, and P2Y4 used in this study were purchased from Applied Biosystems and experiments for validation of amplification efficiency were performed for each TaqMan probe set [28 (link), 29 (link)]. PCR thermocycling parameters were 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Each sample was run in duplicate and was normalized with the expression of 36B4. The fold induction (FI) was determined in a ΔΔCt based fold-change calculations (relative quantity, RQ, is 2^−ΔΔCt).

For RNA extraction, S. aureus was cultured overnight in Luria–Bertani (LB) broth at 37.0 °C and shaking at 120 rpm with and without sub-inhibitory concentrations of 0.25 µg/ml for Fe16, AMP, and Fe16 + AMP. The RNA extraction was performed using TRIzol reagent® (TRIzol reagent, Invitrogen, Carlsbad, CA) according to the manufacturer instructions. The isolated RNA was purified by ethanol RNA/DNA precipitation and reverse transcription was performed with the transcriptor first strand cDNA synthesis kit for RT-PCR (Roche, Mannheim, Germany) based on the manufacturer instructions using 500.0 ng RNA.

Total RNA was extracted from frozen LV tissue with TRIzol reagent (Invitrogen) and 1 μg was reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit for RT-PCR (Roche). Real-time PCR was performed on a MyiQ Real-Time PCR System (Bio-Rad) using Taqman Gene Expression Assays (Applied Biosystem). PCR thermocycling parameters were 50°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Expression of type I collagen was measured using the Quanti SYBR Green RT protocol (Roche). Melting curve data were collected to check the PCR specificity. Each cDNA was amplified in triplicate and the corresponding sample without reverse transcriptase (no-RT sample) was included as a negative control. Gene expression levels were normalized to that of 36B4 mRNA. Replicates were then averaged, and fold induction was determined using a ^ΔΔCt-based fold-change calculation. The primers used were type I collagen (forward primer 5′-AAT GGC ACG GCT GTG TGC GA-3′, reverse primer 5′- AGC ACT CGC CCT CCC GTC TT-3′) and 36B4 (forward primer 5′-AGA TGC AGC AGA TCC GCA T-3′), (reverse primer 5′-GTT CTT GCC CAT CAG CAC C-3′).