Superscript 3 enzyme

Total RNA from pancreas was extracted using TRIzol Reagent, and reverse transcription performed with random primers and Superscript III enzyme (all from Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed in a 7900 HT-Fast real-time PCR (Life Technologies) system with Taqman Universal PCR Master Mix using Taqman assays (Life Technologies) or probes from the Universal Probe Library (Roche, Mannheim, Germany).

RNA was extracted from cell pellets using the RNeasy Micro Plus kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Reverse transcription was performed on 1μg of purified RNA using a Superscript III enzyme (Life Technologies) following manufacturer’s instructions. Primers for PCR and qPCR (Supplementary Table 1) were either sourced from the literature or designed using Primer Blast (NCBI), synthesised (GeneWorks, Hindmarsh, SA, Australia) and validated for species specificity with Primer Blast (NCBI). qPCR was performed using QuantiTectTM SYBR Green master mix (Qiagen) on a Rotor-Gene thermocycler (Corbett Research, NSW, Australia) with reaction parameters: 15 minutes at 95 °C, then cycling of 10 seconds at 95 °C, 20 seconds at 57–60 °C, 30 seconds at 72 °C; for 45 cycles followed by a melt phase. Relative gene expression levels were calculated using the comparative quantitation method available in the Rotor-Gene Software (Corbett Research) and normalised against housekeeper genes GAPDH, ACTB, and CYCA validated using the geNorm algorithm (M value < 1.5).

The primers for quantitative real time PCR are those used for the control of the BAC specificity as they have been chosen in exons (Table 1). The β-2-Microglobulin was chosen as the internal reference gene. Resting and activated macrophage samples of 5 pig and 5 human donors were used. Total RNA was isolated with NucleoSpin® RNA kit (Macherey Nagel) according to the manufacturer instruction. The integrity and quantification of the RNA samples were assessed using an Agilent Bioanalyzer 2100 with a RNA 6000 Nano Lab. RNA (0.5 μg) with a RIN score between 8 and 10 were reverse transcribed using random primers and Superscript III enzyme (Life Technologies). The resulting cDNA samples were diluted 1/25. QPCR was performed in duplicate with 2 μl of the dilution in a final volume of 8 μl using SYBR™ Green PCR Master Mix (Applied biosystems) on a QuantStudio6 real-time PCR System (Thermo Fisher Scientific) as previously described [56 ]. The efficiency of real-time PCR amplification was calculated for each primer pair using six serial dilution points from a cDNA pool including pure resting and activated cDNA samples from resting and activated samples (1:5; 1:10; 1:20; 1:40; 1:80; 1: 160). After determination of the threshold cycle (Ct) for each sample, the PFAFFL method was applied to calculate the relative expression of each gene [57 ] using the cDNA pool as calibrator sample.

RNA of differentiated cells was isolated using the RNeasy Micro Kit (Qiagen) following the manufacturer’s instructions and frozen at -80°C. Then 1 µg total RNA was reversed-transcribed by Superscript III enzyme (Life Technologies) according to the manufacturers’ instructions (see supplementary Materials). Subsequently, both target and housekeeping gene expression levels were analyzed by TaqMan qRT-PCR instrument (CFX384 real time system, Bio-Rad) using the iScript one-step RTPCR kit for probes (Bio-Rad) (see supplementary Materials).

RT reactions were carried out with 70 ng of total RNA with the use of Superscript III enzyme (Life Technologies) and gene-specific primers or oligo(dT) primers.

Total RNA was isolated from the embryonic whisker pad using RNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was extracted, and 250 ng of each sample were reverse transcribed using the Superscript III enzyme and random primers (Life Technologies, Carlsbad, United States).

Total RNA was isolated from cultures of Synechocystis strains at OD₇₃₀ ~ 0.7 as previously described (Mohamed and Jansson 1989 (link)). Turbo DNA-free Kit (Thermo Fisher Scientific) was used to remove carried over genomic DNA per manufacturer’s instructions. The reverse transcription reactions were carried out using Superscript III enzyme (Life Technologies) and random hexamers (New England BioLabs). The cDNA molecules were then used as templates for PCR, employing the same set of primers used to amplify the heterologous genes. As negative controls, DNase-treated RNA molecules were used as templates, and the same primer pairs were employed. petA (sll1317) was used as the positive control as described (Ranade et al. 2015 (link)). For longer templates such as araFGH and araBAD gene sets, the SuperScript One-Step RT-PCR system for long templates (Thermo Fisher Scientific) was used per manufacturer’s instructions. PCR products were analyzed on 0.8% agarose gel.