Revertaid rt reverse transcription kit

Total RNA was extracted from the heart tissue (infarct border zone) with TRIzol Reagent (TRIzol^TM LS Reagent, #10296010, Invitrogen) according to the manufacturer’s protocol and was converted into cDNA with a RevertAid RT Reverse Transcription Kit (RevertAid RT Reverse Transcription Kit, K1691, Thermo Scientific). Real-time polymerase chain reaction (RT-PCR) was performed with a LightCycler 480^® System (LightCycler 480^® Instrument, Roche) by using SYBR Green I Master (LightCycler^®480 SYBR Green I Master, #04707516001, Roche). The relative gene expressions were analyzed by the formula 2^−ΔΔCT method. The primer sequences applied in the procedure are as follows:

Total RNA was isolated from cells according to a single-step method that was described previously by Chomczynski and Sacchi [20 (link)] and Reverse Transcription Polymerase Chain Reaction (RT-PCR) was performed as described elsewhere [14 ]. RNA quality and quantity were determined with spectrophotometry (Epoch, BioTek, Winooski, VT, USA). 150 ng of total RNA was used for genomic DNA digestion and cDNA synthesis. Digestion of genomic DNA was performed using DNase I, RNAase-free (Life Technologies) while complementary DNA (cDNA) synthesis were performed using RevertAid RT Reverse Transcription Kit (Life Technologies). For each reaction, 150 ng of total RNA was used. Genomic DNA digestion and cDNA synthesis were performed using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The qRT-PCR reactions were carried out using a SensiFAST SYBR Green Kit (Bioline, London, UK). Primer concentration equalled 0.5 μM. Sequences of the primers that were used in reactions are listed in Table 1. All qRT-PCR reactions were conducted with CFX Connect™ Real-Time PCR Detection System (Bio-Rad). Relative gene expression analysis (Qn) were evaluated in relation to the GAPDH as a housekeeping gene using ΔΔCt method. Moreover, the ratio of MFN/FIS expression was determined by dividing ΔΔCt of genes.

Total RNA was isolated using TRIsure (Bioline, Luckenwalde, Germany) as manufacturer’s protocol. For cDNA synthesis, total RNA was reverse-transcribed using RevertAid RT Reverse Transcription kit (Life Technologies). Real-time PCR assay was performed using LightCycler^® 480 SYBR Green I Master kit (Roche) (for primer sequences see Supplementary Table 3), and ACTB and HPRT were used as housekeeping genes. Taqman microRNA reverse transcription kit and Taqman gene-specific microRNA assays (Applied Biosystems, Weiterstadt, Germany) were used for miRNA quantifications according to manufacturer’s protocol. RNU44 or RNU48 were used as housekeeping genes for miRNA qRT-PCRs. Both mRNA and miRNA data analyzed according to ΔΔC_T method76 (link).