Omniscript reverse transcription kit

Total RNA was extracted from peripheral blood cells using an RNeasy Mini kit (TaKaRa Bio Inc., Japan). Complementary DNAs were generated with an Omniscript Reverse Transcription kit (TaKaRa Bio Inc., Japan), and BANK1 mRNA expression was quantified using the following primers (Invitrogen, Thermo Fisher Scientific Inc., MA, USA): forward: 5’-CAGACCTGCTGCATATTGCT-3’ and reverse: 5’-CTTGCTTGCTATTTCTGCCA-3’. PCR results were normalized to the expression of the housekeeping gene β-actin.

Total RNA from leaves and the pericarp of fruit at various stages during ripening was extracted using Plant RNA Purification Reagent (Invitrogen, Waltham, MA, USA; 12322-012). 1000 ng of total RNA used for first-strand cDNAs with Omniscript Reverse Transcription kit (Takara, Shiga, Japan; RR047). Gene-specific oligonucleotides were designed with Primer Express software (PE-Applied Biosystems, Waltham, MA, USA). RT-qPCR was performed as described in ref. ^{34 (link)}., using the Bio-Rad CFX384 Real-Time PCR System (BIO-RAD, Hercules, CA, USA) with SYBR Green qPCR Mix (Vazyme, Nanjing, China; Q431-02). Three independent biological replicates were used for each sample. The data was analyzed by comparative threshold cycle method (ΔΔCt). Actin (Solyc11g005330) gene was used as internal control.

Total RNA was extracted from the tissue using the reagent box of Total RNA Kit (Invitrogen, Carlsbad, CA, US), according to the manufacturer's instructions. The concentration of RNA was measured by using a spectrophotometer and the purity was ascertained by the A 260/A 280 ratio with a Nanodrop® 8000. Total RNA from each sample was reverse transcribed to cDNA with an Omniscript® Reverse Transcription kit (Takara) with Oligo-dT primers (Takara) according to the manufacturer's instructions and used for RT-PCR. The target fragments were quantified by real-time PCR using a QuantiTectTMSYBR Green® PCR Kit (Roche) with 100 ng of the cDNA template. Each sample was tested in duplicate. The gene expression data were normalized to β-actin expression. The primers used correspond to the rat sequences shown in Table 1; primer design was done using Amplify software (TaKaRa, Nanjing, China). For each real-time PCR assay, the threshold cycle Ct was determined for each reaction. Ct values for each gene of interest were normalized to the housekeeping gene (β-action); PCR amplification efficiencies were taken into account by amplifying various amounts of target cDNA for each reaction. The fold differences in mRNA expression of samples were relative to the internal control sample, which was included in all runs.