Easyscript all in one first strand cdna synthesis supermix kit

Root tissues from WT and Stumpy mutant grown for 20 days in hydroponic solution with CaCl₂ added to 0.5 mM or 10 mM were collected in liquid nitrogen. Total RNA was extracted from roots using an OminiPlant RNA Kit (CWBIO, Jiangsu, China) according to the manufacturer’s instructions, and first-strand cDNA was synthesised using the EasyScript® All-in-One First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). The cDNA was diluted with water in a 1:5 ratio, and 2 μl of diluted cDNA was used for qRT-PCR with the 2xSuperFast Universal SYBR Master Mix (CWBIO) using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The wheat β-ACTIN gene was used as an endogenous control (Paolacci et al. 2009 (link)). Primers used for qRT-PCR are listed in Table S1.

An EasyScript All-in-One First-Strand cDNA Synthesis SuperMix kit (Transgen, Beijing, China) was used for reverse transcription. LncRNAs, novel transcripts, and fusion genes were identified from cDNA amplification; qRT-PCR was performed using the CFX96 Touch Real-Time PCR (Bio-Rad, Hercules, CA, USA) with the SYBR Green Realtime PCR Master Mix (Biorad, CA, USA). The relative gene expression was determined using the ∆∆Ct method. Each sample was repeated in triplicate. Total RNA was self-linked to a circle using a T4 RNA Ligase (Takara, Tokyo, Japan); then, the circular RNA was extracted with chloroform, precipitated in ethanol, and dried. Reverse transcription was performed with the Reverse Transcriptase M-MLV (RNase H-; Takara, Tokyo, Japan) with the CRT-PCRF and CRT-PCRR2 primers (Table S9). PCR products were detected with agarose gel electrophoresis, whereas TA cloning and sequencing were performed after product recovery (Data S1). A one sample t-test was performed on –∆∆Ct for a significant difference comparison.