Reverse transcription system

RNA extraction and quantitative RT-PCR was performed as described in our prior study [7 (link)]. Briefly, total RNA was extracted from 200 mg of seeds using the RNeasy Plant Mini Kit (Qiagen, CA, USA) and treated with an RNase-free DNase I digestion kit (Beijing Aidlab Biotech Company, Beijing, China) to remove any residual genomic DNA. Then 1 μg of RNA was reverse-transcribed to cDNA, using a reverse transcription system (Bio-Rad Laboratories, Inc., Richmond, CA, USA), and the cDNA equivalent to 25 ng of total RNA served as a template for each PCR reaction, carried out using SYBR Green supermix (Bio-Rad Laboratories, Inc., Richmond, CA, USA) with a final concentration of 1.6 mM of each primer. The primer sequences are listed in Table S1. The qRT-PCR experiments were done using a SYBR Premix Ex TaqTM (Takara Biotechnology Co., Ltd., Dalian, China) on the LightCycler 480 machine (Roche Applied, Mannheim, Germany). PCR amplifications of three biological replicates were performed, which also included three distinct technical replicates. A no-template control (i.e., RNase-free water) was included for every qPCR run. Transcript abundance was normalized using the housekeeping gene EF-1α and a given gene’s expression level amount was calculated by the 2^−ΔΔC_T method [44 (link)].

For RNA extraction, TRIzol reagent (Invitrogen, Grand Island, NY, United States) was used to isolate total RNA. The total RNA was reverse transcribed to cDNA using a reverse transcription system (Bio-Rad, Hercules, CA, United States) according to the manufacturer’s instructions. The mRNA expression level of the gene of interest was determined through qRT-PCR using a Bio-Rad CFX96 system with SYBR green. GAPDH expression was used to normalize the expression levels of the target gene. The details of primers used in the study are shown in Supplementary Table 1.

RT-qPCR was performed to analyze the mRNA expression levels of Ext1. TRIzol reagent (Invitrogen) was used to extract the RNA from the 293T-ACE2 cells and then reverse transcribed into cDNA using the Bio-Rad reverse transcription system (#1708840). Bio-Rad SYBR-Green Supermix (#1708880) was used for qPCR analysis. The primer sequences used in this experiment were: Ext1 forward, 5′-GCTCTTGTCTCGCCCTTTTGT-3 and reverse, 5′-GGTGCAAGCCATTCCTACC-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-GTATTGGGCGCCTGGTCACC-3′ and reverse, 5′-CGGGAAGATGGTGATGG-3′. The PCR-amplified mRNA was quantified and the results were normalized against GAPDH expression. The 2^–ΔΔCq method was used to calculate the relative mRNA expression level.