Hiscript 2 q select rt supermix for qpcr gdna wiper

One-week-old seedlings grown on 1/2 MS agar medium were treated at 37°C or 22°C in growth chambers for the indicated time. Harvested plant tissue was ground in liquid nitrogen to a fine powder, and total RNA was extracted from 100 mg of tissue using TRIzol Reagent (CWBio, Taizhou, Jiangsu, China) according to the manual provided by the manufacturer. First-strand cDNA was synthesized from 1 μg of total RNA using HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Nanjing, Jiangsu, China). Quantitative PCR (qPCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co.) on a C1000 Touch Thermal Cycler CFX384 (Bio-Rad Hercules, CA, United States). The relative abundance of each transcript was determined by the comparative threshold cycle method (Schmittgen and Livak, 2008 (link)), using UBC30 as an equal loading control. The gene-specific primers used are listed in Supplementary Table 4.

A total RNA extract of NB cells was prepared using TRIzol reagent (15596026, Invitrogen, USA). The reverse transcription of RNA was performed using the HiScript® II Q Select RT SuperMix for qPCR (+gDNA wiper) (R233-01, Vazyme, China) as the provided protocol. The cDNA was used for qualitative real-time PCR using the Hieff® qPCR SYBR Green Master Mix (11202ES03, YEASEN, China). The usage of primers for RT-qPCR were as follows: GAPDH forward, 5-TGCACCACCAACTGCTTAG-3, reverse, 5-GATGCAGGGATGATGTTC-3; METTL14 forward, 5-AGAGAAACTGGCATCACTGCT-3, reverse, 5-TCGTAAACACACTCTTCCAAGG-3; YTHDF1 forward, 5-TGTGGAATGAGGGACCGTTG-3, reverse 5-GGATCCTCTACAAGGGCACG-3; YTHDF2 forward 5-GCGACACATTCGCCTAGAGA-3, reverse 5-GGAAGTGGTGTGCTTGTAGC-3; YTHDF3 forward 5-TTTTCATCAGCGCATCTGCC-3, reverse 5-TCTGTGCCTTAGCTAGGATGC; YY1 forward 5-ACGGCTTCGAGGATCAGATTC-3, reverse 5-TGACCAGCGTTTGTTCAATGT-3; YWHAH forward 5-GACATGGCCTCCGCTATGAAG-3, reverse 5-ATGCTGCTAATGACCCTCCAG-3.

The RNA sequencing results were verified by RT-qPCR (Bio-Rad CFX 96™, Hercules, CA, USA). For that, 26 genes were selected based on their function classification and differential expression in the RNA-Seq results. These included the cas operon, lsr operon, and T3SS genes. For RT-qPCR, briefly, one microgram of total RNA was reverse transcribed into complementary DNA (cDNA) using the HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The cDNA was amplified by RT-qPCR using SYBR Green Real time PCR Master Mix (Takara, Japan). Relative quantification of gene expression was calculated using the 2^−ΔΔCt method and normalized to reference gene 16S rRNA in each sample, in which cas3 WT was used as a control. The primers used in RT-qPCR are listed in the Table S5.

Briefly, total RNA was extracted from gastric cancer tissues or cells by Trizol (Invitrogen). HiScript II Q Select RT SuperMix for qPCR (+gDNA Wiper) (Vazyme, #R233) was used to reverse RNA samples to cDNA. The gDNA wiper Mix in this kit was utilized to remove of genomic DNA. No RT Control Mix in this kit was utilized to confirm the complete removal genomic DNA. ChamQ SYBR qPCR Master Mix (Vazyme, #Q311) was used to detect RNA expression levels by qPCR with a Bio‐Rad CFX 96 Real‐Time System (Bio‐Rad). Primer sequences were provided in Table S1. The specificity of the PIN1P1 and PIN1 primers was verified by the NCBI Primer‐BLAST tool and Sanger‐based sequencing of the qPCR products (Figure S1).

Total RNA was extracted using TRIzol reagent. One microgram of RNA was used to synthesize cDNA with HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) from Vazyme (Code No. R233–01) following specific protocols. For qPCR, 1 µL of cDNA was amplified using Taq Pro Universal SYBR qPCR Master Mix from Vazyme (Code No. Q712–02). β-actin served as the endogenous control. Primer sequences for qRT-PCR were as follows: β-actin: Forward: 5′- CATGTACGTTGCTATCCAGGC-3′, Reverse: 5′- CTCCTTAATGTCACGCACGAT-3′; for LDHA: Forward: 5′- TGGAGATTCCAGTGTGCCTG-3′; Reverse: 5′- TAGCCCAGGATGTGTAGCCT-3′; for LDHB: Forward: 5′- ACCAGTTGCGGAAGAAGAGG-3′; Reverse: 5′- CTCCCATGCTGCAGATCCAT-3′.

RNA-seq data for 16 different genes was validated by real-time quantitative PCR (qPCR). The primers of selected genes were designed using Primer Priemer 5 software (PREMIER Biosoft, Palo Alto, CA, USA) and synthesized by Sangon. The primer pair SmActin-F and SmActin-R was used to amplify an eggplant actin fragment as a control for normalizing the starting amounts of cDNA. The sequences of primers used in this study are listed in Supplementary Table S4. The first-strand cDNA was synthesized using 1 μg total RNA by HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) in 10 μL of reaction mixture. The quantitative PCRs were performed according to the manufacturer's instructions using the DyNAmo™ Flash SYBR Green qPCR Kit (Tiangen Biotech, Beijing, China) in an ABI Prism StepOnePlus real-time thermal cycler (Applied Biosystems, Carlsbad, CA, USA). The amplification was performed as follows: 10 min at 94°C followed by 40 cycles of 94°C for 20 s and 60°C for 60 s. A melting curve was generated to ensure product uniformity. Gene expression was evaluated by the 2^_△△Ct method [26 (link)]. The expression of genes was related to the SmActin expression. The correlation between expression profiles of selected genes obtained from real-time PCR and RNA-seq data based on log2 RPKM values was determined using MS Excel 3.7.