High capacity cdna reverse transcription kits with random primers

Total RNA was prepared using RNA Bee (AMS Biotechnology Ltd) with 1 ml per 4 × 10⁶ cardiomyocytes or 10–15 mg mouse heart powder. RNA was prepared according to the manufacturer's instructions, dissolved in nuclease-free water and purity assessed from the A₂₆₀/A₂₈₀ measured using an Implen NanoPhotometer (values of 1.8–2.1 were considered acceptable). RNA concentrations were determined from the A₂₆₀ values. Quantitative PCR (qPCR) was performed as previously described [28 (link)]. Total RNA (1 µg) was reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kits with random primers (Applied Biosystems) according to the manufacturer's instructions. qPCR was performed using an ABI Real-Time PCR 7500 system (Applied Biosystems). Optical 96-well reaction plates were used with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc.) according to the manufacturer's instructions. Primers were from PrimerDesign, Eurofins Genomics or Thermo Fisher Scientific (see Supplementary Table S4). GAPDH was used as the reference gene for the study. Results were normalised to GAPDH, and relative quantification was obtained using the ΔCt (threshold cycle) method; relative expression was calculated as 2^−ΔΔCt, and normalised to vehicle or time 0.

Mouse heart powders were weighed into safelock Eppendorf tubes and kept on dry ice. RNA Bee (AMS Biotechnology Ltd) was added (1 ml per 10–15 mg) and the samples homogenized on ice using a pestle. RNA was prepared according to the manufacturer’s instructions and dissolved in nuclease-free water. The purity was assessed from the A₂₆₀/A₂₈₀ measured using an Implen NanoPhotometer (values were 1.8–2.0) and concentrations determined from the A₂₆₀. Quantitative PCR (qPCR) analysis was performed as described in [31 (link)]. Total RNA (1 µg) was reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kits with random primers (Applied Biosystems). qPCR was performed using an ABI Real-Time PCR 7500 system (Applied Biosystems) using 1/40 of the cDNA produced. Optical 96-well reaction plates were used with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories Inc.) according to the manufacturer’s instructions. See Supplementary Table S3 for primer sequences. Results were normalized to Gapdh, and relative quantification was obtained using the ΔCt (threshold cycle) method; relative expression was calculated as 2^−ΔΔCt, and normalized as indicated in the figure legends.

Quick-frozen tissue samples of liver were powdered with cold mortar and pestle, and ~100 mg was used for the isolation of RNA. Total RNA was extracted by the guanidine thiocyanate method and mRNA purified using PureLink RNA Mini Kits (Invitrogen) according to the manufacturer’s instruction. Total RNA was reverse transcribed in a final volume of 20 μL using high capacity cDNA reverse transcription kits with random primers (Applied Biosystems, Foster City, CA) as described by the manufacturer. Reverse transcribed samples were stored at -20°C.