Superscript 3 rnase h

Quantitative real-time (RT) PCR analysis was performed using an established procedure [19 (link)]. Total RNA was extracted using QIAGEN extraction kit and reverse transcription was performed using SuperScript III RNase H (Invitrogen, Waltham, MA, USA). Quantitative RT-PCR was performed using SYBR Green I and a LightCycler (Roche Diagnostics, Castle Hill, Australia). For normalized gene expression, 18S was used as the reference gene in all experiments. Table S1 lists the primers used for RT-PCR analysis.

The mRNA expression of growth hormone, prolactin, deiodinase 1 and 2, thyroid stimulating hormone, and thyroid-releasing hormone was analysed in the pituitary of female adult trouts from cobalt blue (n = 4; BW = 880±28.3 g; SL = 35.2±0.5 cm; GSI = 1.42±0.56) and wild type phenotypes (n = 4; BW = 925±156.1 g; SL = 36.2±2.4 cm; GSI = 1.69±1.53). Samples were fixed in RNAlater and stored at -80 °C until processing. Total RNA was extracted from pituitary using 1 ml of TRIZOL Reagent (Invitrogen) following the manufacturer’s instructions. RNA samples (1 μg) were treated with Deoxyribonuclease I Amplification Grade (Invitrogen) and reverse-transcribed using SuperScript III RNase H- (Invitrogen) with oligo(dT)12-18, following the manufacturer’s instructions. Primer sequences, amplicon size, and GenBank accession numbers are described in S2 Table. Real Time PCR was performed in 15 μl reaction volumes containing 2x Power SYBR^® Green PCR Master Mix (ABI), 1 μl of first strand cDNA (approximately 25 ng) and 5 pmol of each primer in a Step One Real Time PCR System (Applied Biosystems, Foster City, USA), using standard program. Transcript abundance was quantified using the Standard Curve Method with four dilution points and normalized against respective β-actin values.

RNA isolation and RT-PCR were performed as previously described [12 (link)]. Briefly, ES cells or EBs were collected and immediately suspended in Tri Reagent (Molecular Research Center), and RNA was extracted according to the manufacturer’s instructions. Total RNA (2 μg) and oligo-d(T) primers (500 ng; see S2 Table for sequences) were used to synthesize cDNAs at 42°C for 90 min using Superscript III RNase H reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time RT-PCR reactions were performed using the Chromo4 real-time detection system (Bio-Rad).