Rnaseout recombinant rnase inhibitor

RNA was extracted from frozen left ventricle and soleus using a Qiagen RNeasy Fibrous Tissue Mini kit according to manufacturer’s instructions. 5 μM random hexamers and 1 mM deoxynucleotide triphosphates were combined with 1 μg RNA in a 10 μl solution. Primer annealing was initiated by incubation at 65°C for 5 min in a Veriti 96-well Thermocycler (Applied Biosystems). Samples were combined with 200 U SuperScript III reverse transcriptase, 40 U RNaseOUT recombinant RNase inhibitor, 200 mmol dithiothreitol, 100 mmol MgCl₂ and 1 × reverse transcriptase buffer (Life Technologies) in a reaction volume of 20 μl. Following initial incubation in the Thermocycler (10 min, 25°C), complementary DNA was synthesised (50 min, 50°C), after which the reaction was terminated (5 min, 85°C). Real-time quantitative PCR was performed in triplicates in 96-well plates on a StepOne Plus detection system (Applied Biosciences) with an initial incubation period (10 min at 95°C), then 40 cycles of elongation (15 s at 95°C) and cooling (1 min at 60°C). Taqman probe/primer assay mix (Life Technologies) for Ppara, Ucp3, Acadm and Cpt1b were used in Taqman Universal PCR Master Mix. Expression levels of targets were normalised to Hprt by the ΔCT method.

Pre-mRNA templates were transcribed using the MEGAscript T3 Transcription Kit according to the manufacturer’s instructions (Thermo Fisher No. AM1338). In vitro splicing reactions were assembled in 25 μl volumes, each containing 10 μl of splicing extract, 5 μl of 5x splicing buffer (12.5 mM MgCl₂, 15% PEG 8000, 300 mM KH₂PO₄ at pH 7.2, and 30 μl nuclease-free water), 5 μl pre-mRNA template from in vitro transcription, 0.5 μl 200 mM ATP, 1 μl RNaseOUT Recombinant RNase Inhibitor (Life Technologies No. 10777–019), and 3.5 μl nuclease-free water. For reactions involving the addition of regulatory element RNA, this RNA was added to reactions in lieu of water to the final concentrations indicated in the figures. Reactions were incubated at 25°C for 40 minutes and then stopped by the addition of 75 μl nuclease-free water and 100 μl of RNA buffer followed immediately by phenol-chloroform RNA extraction. The extracted RNA was DNase-treated and then used for RT-PCR analysis (see below). Equal amounts of DNase-treated RNA were used for all samples for the cDNA synthesis reactions.

The cDNA was synthesized from DNase-treated RNA (1,000 ng) using 0.5 μg oligo-dT_12-18, 100 U Superscript III reverse transcriptase, and 20 U RNaseOUT recombinant RNase inhibitor (all from Thermo Fischer Scientific) in a total volume of 20 μl for 2 h at 42°C followed by heat inactivation for 15 min at 70°C. Quantitative PCR with EvaGreen QPCR mix II (Bio&SELL) was performed with 1:10 diluted cDNA. Primers were used at a final concentration of 500 nM. Target gene expression was calculated using the ΔΔCT method (79 (link)), with normalization to the housekeeping genes CgACT1 for C. glabrata or ScACT1 for S. cerevisiae. For mtDNA quantification, yeast DNA was extracted following the Harju et al. protocol (80 (link)), and 100 ng was the reaction concentration. ScCOX3 and CgCOX3 were used as mitochondrial target genes and CgACT1 or ScACT1 as housekeeping genes. All experiments were done in independent biological triplicates on different days and are shown as the mean with standard deviation (SD) for each time point.

PBMCs were incubated with 2 ug/mL tunicamycin in RPMI 1640 for 4 h. RNA was isolated using RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. For reverse transcription-quantitative PCR (RT-qPCR), isolated RNA (500 ng) was treated with DNase I (Fermentas) and subsequently transcribed into cDNA using 0.5 µg Oligo(dT)12–18 Primer, 200 U Superscript™ III Reverse Transcriptase and 40 U RNaseOUT™ Recombinant RNase Inhibitor (Thermo Fischer Scientific). RT-qPCR was performed with GoTaq® qPCR Master Mix (Promega) in a CFX96 thermocycler (Bio-Rad) and the expression levels were normalized against β-actin. All the primers used are listed in Supplementary Table 2.

RT-PCR was conducted using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA). The RNA molecules obtained ex vivo were used as templates. The synthesis of the cDNA was carried out with the use of 2.5 μM random hexamer primers, 0.5 mM dNTP each, and 1 ng of the RNA template. The reaction mix was incubated at 65 °C for 5 min and on ice for 1 min. Subsequently, 4 µl of SuperScript IV RT buffer, 1 µl of 5 mM DTT, 0.5 µl of RNaseOUT Recombinant RNase Inhibitor (40 U/µl) (Thermo Fisher Scientific, Waltham, USA), and SuperScript IV Reverse Transcriptase (200 U/µl) (Thermo Fisher Scientific, Waltham, USA) were added. The samples were incubated in a thermocycler at 23 °C for 10 min, 50–55 °C for 10 min, and at 80 °C for 10 min.

For RT-qPCR, 500 ng RNA was treated with DNase (Fermentas) for 30 min at 37°C and reverse transcribed into cDNA using 0.5 μg Oligo(dT)12-18 Primer, 200 U Superscript III Reverse Transcriptase and 40 U RNaseOUT Recombinant RNase Inhibitor (Thermo Fischer Scientific). cDNA (100 ng) was used for RT-qPCR assay, with GoTaq qPCR mastermix (Promega) in a C1000 thermocycler (BioRad, CFX96 Realtime system). The expression levels were normalized to the housekeeping gene ACT1 and expressed as relative to the expression of the target gene at 0 time point (log₂ fold change). Gene expression analysis was carried out in triplicates for each sample and primer pair. All the primers used can be found in Table 1.