Mint universal cdna synthesis kit

Total RNA was extracted from 30 mg of liver or white skeletal muscle using the RNeasy tissue and RNeasy fibrous tissue, respectively, mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration and purity was determined spectrophotometrically at 260/280 nm using Nanodrop ND-1000 (Thermo Fischer Scientific, Waltham, MA, USA). RNA integrity was determined with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only samples with RNA Integrity Number (RIN) > 9.2 were used for subsequent studies. Pools consisting of 1 μg of total RNA isolated from 6 individuals per condition (fasting and feeding with diets HLL, MHL, MLH, LHH and LLH; 36 fish and RNA samples in total) were used to construct liver and white skeletal muscle cDNA libraries. The dsDNA synthesis was performed using a MINT-Universal cDNA synthesis kit (Evrogen, Moscow, Russia). To increase the presence of rare transcripts, the cDNAs libraries were normalised using TRIMMER cDNA normalization kit (Evrogen, Moscow, Russia) following manufacturer’s instructions. Sequencing of cDNAs libraries was performed using a GS FLX 454 platform (Roche, Basel, Switzerland) at the CCiTUB of the Universitat de Barcelona (Barcelona, Spain).

Total RNA was isolated from Caco-2 cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions after 4 or 24 h of stimulation and cell washing by RPMI-1640 medium. The quality and quantity of total RNA were determined by the A260/280 ratio using NanoPhotometer NP80 (Implen GmbH, München, Germany) as well as agarose gel electrophoresis (Supporting Materials, Figure S3). RNase inhibitor (Evrogen, Moscow, Russia) was added to the final preparation of total RNA. Two μg of total RNA was taken to synthesize the cDNA using MINT-Universal cDNA synthesis kit (Evrogen, Moscow, Russia) containing MMLV-based reverse transcriptase following the manufacturer’s instructions.

Equal amounts of extracted RNA from different seed maturation stages were pooled and used for cDNA library construction. To purify mRNA from 5 μg total RNA, the mRNA-Only Eukaryotic mRNA Isolation Kit (Epicentre) was used by applying exonuclease digestion followed by LiCl precipitation. One μg mRNA was used for the synthesis of the first-strand cDNA by the Mint-Universal cDNA Synthesis Kit (Evrogen). The Trimmer Kit (Evrogen) was used for normalization reaction using 800 ng amplified cDNA, which was re-amplified by 18 cycles.