Trizol reagent (Vazyme) was used to extract total RNA of ccRCC tumor samples or cells following manufacturer’s instructions. Synthesis of complementary DNA (cDNA) was done using the HiScript® II RT SuperMix (Vazyme). StepOnePlus (Life Technologies) apparatus was applied to carry out quantitative real-time PCR (qPCR). The primers are listed in Table S3 . Gene-specific data were normalized to β2-microglobulin and are calculated as relative expression compared to DMSO or control group using the ΔΔCT method.
Hiscript 2 rt supermix
Manufactured by Vazyme
Sourced in China
HiScript II RT SuperMix is a reverse transcription reagent that converts RNA into complementary DNA (cDNA). It is designed for efficient and reliable reverse transcription of various RNA templates, including mRNA, total RNA, and viral RNA.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (3 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
Trizol, by Takara Bio (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr qpcr master mix, by Vazyme (1 mentions)
Pcr master mix, by Vazyme (1 mentions)
Sybr green master mix, by Novogene (1 mentions)
Transstart probe qpcr supermix, by Transgene (1 mentions)
All in onetm mirna qpcr detection kit, by GeneCopoeia (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Rna extraction kit, by Vazyme (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Roche (1 mentions)
Lightcycle480 2 real time pcr detection system, by Roche (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
16 protocols using hiscript 2 rt supermix
1
Extraction and Quantification of ccRCC RNA
2
RNA Extraction and CHRM3 Quantification
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RNA was extracted using TRIzol® reagent (cat 15596026CN, Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription of RNA was done using HiScript II RT SuperMix (cat R223-01, Vazyme, Nanjing, China). Forward primer for CHRM3 was 5′-CGTGGCACCTGGTCTCTTTC-3′ and reverse primer for CHRM3 was 5′-TTCCAGGTAGGAGCATCAAACC-3′.
3
RNA Extraction and qRT-PCR Protocol
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Cells were lysed, and total RNA extracted using TRIzol (TaKaRa) according to the manufacturer’s instructions. cDNA was generated by HiScript II RT SuperMix (Vazyme). Quantitative real-time PCR was performed using SYBR qPCR Master Mix (Vazyme) and QuantStudio 5 real-time PCR system (Applied Biosystems). The primer sequences are provided in S1 Table .
4
Multiplex PCR Detection of Enteric Pathogens
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Intestinal contents were collected from piglets with watery diarrhea and vomiting in the western provinces of China (Gansu, Qinghai, Ningxia, Shanxi, and Chongqing) between 2015 and 2018. The contents were diluted with phosphate-buffered saline (PBS) at a ratio of 1:100 for RNA extraction with RNAiso Plus (Takara, Japan) according to the manufacturer’s instructions. The extracted RNA was then reverse transcribed using HiScript II RT SuperMix (Vazyme, China). Briefly, a mixture of 4 μL of gDNA wiper Mix, 1 μg of RNA, and nuclease‐free water to 16 μl was incubated at 42°C for 2 min, after which 5 μl of HiScript II RT SuperMix was added and the mixture was incubated at 50°C for 15 min and 85°C for 5 s. Detection of pathogens was performed by a multiplex PCR method developed previously in our laboratory [21 (link)]. Briefly, 5 μl of cDNA was used as a template and mixed with the specific primer pair, 12.5 μl of 2× PCR master mix (Vazyme, China), and nuclease‐free water to 25 μl. The reaction was carried out under the following conditions: 95 °C for 3 min, followed by 35 cycles of 15 s at 95°C, 15 s at 52°C, and 30 s at 72°C, and final extension at 72°C for 5 min. The PCR products were then analyzed by agarose gel electrophoresis.
5
Quantifying Host Response to PDCoV
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To examine the host response to PDCoV infection, RNA was extracted from different tissues using RNAiso Plus (Takara, Japan) and then reverse transcribed into cDNA using HiScript II RT SuperMix (Vazyme, China). Real-time PCR analysis was employed for detection with SYBR Green Master Mix (Novogene, China) in a Bio-Rad CFX96 system. The reaction mixtures were incubated at 94°C for 30 s, followed by 40 cycles at 94°C for 5 s and 60°C for 30 s. GAPDH served as an internal control for normalization. The relative expression levels of target genes were calculated by the 2−ΔΔCt method. The viral load was measured using TransStart Probe qPCR SuperMix (Transgen, China). Briefly, the mixtures were incubated at 94°C for 30 s, followed by 40 cycles at 94°C for 5 s and 60°C for 30 s. All primers and probes are listed in Table 3 .
6
Total RNA Isolation and qPCR Analysis
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Total RNA was isolated from ovary tissue or cultured cells and extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The concentration of total RNA was measured by Ultraviolet Spectrophotometer at 260 nm (Thermo Fisher Scientific, Inc.). cDNA was synthesized from 1 µg total RNA using an All-in-one TM miRNA qPCR Detection kit (GeneCopoeia, Inc.) or from 500 ng total RNA using a HiScript II RT SuperMix according to the manufacturer's instructions (Vazyme Biotech Co., Ltd.). qPCR analysis was performed using AceQ qPCR SYBR Green master mix (Vazyme Biotech Co., Ltd.) on an ABI ViiA 7 Real-Time PCR system (ABI, USA). The qPCR was performed under the following conditions: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 1 min. U6 snRNA was used as an internal control for miR-378d and GAPDH was used as an internal control for other genes. Experimental data were analyzed using a relative quantification method (2−∆∆Cq) (30 (link)). All experiments were repeated three times. The RT-qPCR primers are described in detail in Table II .
7
Hypothalamic Gene Expression Analysis
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Mice were euthanized in a CO2 chamber, and hypothalamic tissue was collected in a test tube. Total RNA was extracted from the entire hypothalamic tissue with TRIzol Reagent (Invitrogen). cDNA was synthesized using HiScript II RT SuperMix (Vazyme). Quantitative PCR was performed with ChamQTM Universal SYBR qPCR Master Mix (Vazyme). Messenger RNA expression levels of Pomc, Agrp, Npy, Socs3, and LepR were measured and normalized to the internal control genes (β‐actin).
8
Real-time qPCR Analysis of Antifungal-Treated Biofilms
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RNA-seq data were verified by real-time quantitative PCR (RT-qPCR) analysis. Control and treated biofilms under antifungal agents for 24 h at the concentration of SMIC50 were harvested and frozen in liquid nitrogen for RNA extraction. Extraction of total RNA was carried out using an RNA extraction Kit (Vazyme, Nanjing, China). The integrity of the RNA of each group was measured using nanodrop (NanoDrop One, Thermo Fisher Scientific Inc., Waltham, MA, USA), and then the extracted RNA was submitted to be reversely transcribed into cDNA using HiScript II RT SuperMix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The cDNA was used to analyze the expressions of ROS-related genes and autophagy-related genes, which was performed on a QuantStudio™ 7 Flex (Thermo Fisher Scientific Inc., Waltham, MA, USA) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The relative expressions of genes were determined using the 2−ΔΔCt method [26 (link)]. β-actin served as an internal reference gene. The primers and sequences are listed in Table 1 . Three biological replicates were prepared for each sample and the test was repeated three times.
9
Quantitative Real-Time PCR Analysis
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Total RNA from the human samples or the cell lines was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse-transcribed according to the manufacturer's protocol using HiScript®II RT SuperMix (Vazyme Biotech Co., Ltd.). qPCR was performed in a Roche LightCycle480 II Real-Time PCR Detection System using SYBR Premix Ex Taq (cat. no. RR420A; Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 10 min, followed by 45 cycles at 92°C for 15 sec and 72°C for 5 min. All the measurements were performed in triplicate. The sequences of the primers used in the present study are listed in Table SI . Quantitative mRNA data were normalized and presented as a ratio to GAPDH, calculated using the 2−ΔΔCq method (49 (link)).
10
Quantitative RNA Expression Analysis
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RNA extraction from cells and tissues was conducted using the TRIZol reagent (TIANGEN). Subsequently, the purified RNA underwent reverse transcription using the HiScript II RT SuperMix (Vazyme, R223-01). The expression levels of target genes were assessed using SYBR Green qMix (Vazyme, Q311). The RT-qPCR data of this study represented the relative expression levels of target mRNAs normalized to the expression of Gapdh. The primers utilized in this investigation are listed in Supplementary Table 8
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