Total RNA was extracted using TRIzol reagent (Life), 5× gDNA digester (Yeasen) was used to erase genomic DNA along with the procedure carried out at 42°C for 2 min before adding 4xHifair® III SuperMix plus (Yeasen) followed by 37°C for 15 min and 85°C for 5 s for reverse transcription to synthesize cDNA. Quantitative real‐time PCR was performed using a Roche instrument and the SYBR Green PCR master mix (Yeasen); the procedures were as follows: 95°C for 5 min, 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. GAPDH was used as the internal control. Primer sequences are listed in Table S2 .
Gdna digester
Manufactured by Yeasen
Sourced in China
The GDNA digester is a laboratory instrument designed to extract and purify genomic DNA (gDNA) from biological samples. It utilizes enzymatic digestion and chemical lysis to efficiently release and isolate gDNA, which can then be used for various downstream applications such as genetic analysis and sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Yeasen (3 mentions)
4xhifair 3 supermix plus, by Yeasen (2 mentions)
Sybr green pcr master mix, by Yeasen (2 mentions)
Quantstudio 1 real time pcr detection system, by Thermo Fisher Scientific (2 mentions)
Hifair 2 1st strand cdna synthesis kit, by Yeasen (2 mentions)
Hieff qpcr sybr green master mix, by Yeasen (2 mentions)
Rnaiso plus reagent, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Axyprep body fluid viral dna rna miniprep kit, by Corning (1 mentions)
Np80 uv vis spectrophotometer, by Implen (1 mentions)
Hifair 2 1st stand cdna synthesis supermix, by Yeasen (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Trizol reagent, by Yeasen (1 mentions)
Hifair 3 1st strand cdna synthesis supermix, by Yeasen (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Total rna isolation reagent, by Biosharp (1 mentions)
Hifair 2 1st strand cdna synthesis supermix, by Yeasen (1 mentions)
Lightcycler real time pcr system, by Roche (1 mentions)
Sybr green master mix, by Yeasen (1 mentions)
11 protocols using gdna digester
1
Quantification of Gene Expression
2
Quantification of FASN Expression
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TRIzol reagent (Life Technologies, USA) was used to extract total RNA from cell lines. Genomic DNA was then erased using a 5 × gDNA digester (Yeasen) at 42 °C for 2 min. Next, 4 x Hifair ® III SuperMix plus (Yeasen) was added to reversely transcribe RNA into complementary DNA at 37 °C for 15 min and 85 °C for 5 s. qRT-PCR was carried out using SYBR Green PCR master mix (Yeasen). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control for FASN. The primers used in the present study were: FASN-forward, 5'-AAGGACCTGTCTAGGTTTGATGC-3', FASN-reverse, 5'-TGGCTTCATAGGTGACTTCCA-3'; GAPDH-forward, 5'-ATCACCATCTTCCAGGAGCGA-3', GAPDH-reverse, 5'-CCTTCTCCATGGTGGTGAAGAC-3'. Data were analyzed using the 2-△△ct method and visualized using GraphPad Prism 9 software.
3
SARS-CoV-2 RNA Quantification by qRT-PCR
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Cell culture supernatants and Vero E6 cells were harvested for RNA extraction using the AxyPrep™ body fluid viral DNA/RNA Miniprep kit (Axygen, Cat No. AP-MN-BF-VNA-250, Hangzhou, Zhejiang, China) and AxyPrep™ multisource total RNA Miniprep kit (Axygene, Cat No. AP-MN-MS-RNA-250G) according to the manufacturer's instructions. Reverse transcription was performed with a Hifair II 1st Strand cDNA synthesis kit with gDNA digester (Yeasen Biotech, Cat:11121ES60, Shanghai, China), and qRT-PCR was performed using QuantStudio 1 Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with Hieff qPCRSYBR Green Master Mix (Yeasen Biotech, Cat:11202ES08, Shanghai, China) or two-step Taqman probe assay. The sequence information of primers used is listed in Supplementary Table 1. And the PCR products were inserted into T vector (Ruibo Xingke Biotech, Beijing, China) to generate the standard plasmid after sequencing confirmation. The standard curve was generated by determination of copy numbers from serially dilutions (103 − 109 copies) of the plasmid. qRT-PCR amplification of SYBR Green method was performed as follows: 95°C for 5 min followed by 40 cycles consisting of 95°C for 10 s, 55°C for 20 s, and 72°C for 31 s. And the Taqman method was performed as follows: 50°C for 2 min, 95°C for 10 min followed by 40 cycles consisting of 95°C for 10 s, 60°C for 1 min.
4
RNA Extraction and cDNA Synthesis
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Total RNA was extracted from three pools of tissues, different stages, and survived induction nymphs using RNAiso Plus (Takara, Shiga, Japan), following the manufacturer’s instructions. DNA contamination was removed by treating RNA extraction products with gDNA digester (Yeasen, Shanghai, China). The quality and concentration of RNA samples were checked spectrophotometrically at 260/280 nm using an NP80 UV–Vis spectrophotometer (IMPLEN, Munich, Germany). One microgram of total RNA was reverse transcribed to first-strand cDNA by using Hifair™ II 1st Stand cDNA Synthesis SuperMix (Yeasen, Shanghai, China).
5
qPCR Analysis of Gene Expression
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Total RNA samples were extracted with the RNAiso plus reagent (Takara, Japan). After digestion with gDNA digester (Yeasen, China), cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Yeasen). The reference gene RP18 was chosen as internal control (Pfaffl et al., 2004 (link); Huggett et al., 2005 (link)). The reaction mixture consisted of 5 µl TB Green Premix Ex Taq II (Tli RNaseH Plus), 1 µl of cDNA, and 0.25 µl of forward and reverse primers (see Supplementary Table S2 ; 10 µM) in a final reaction volume of 10 µl. The qPCR protocol included an initial denaturation step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and 72 °C for 30 s. The relative expression of β-Actin was calculated by the method (Livak and Schmittgen, 2001 ; Shi et al., 2013 (link)). All experiments were repeated in triplicate.
6
Gene Expression Analysis of WFTs
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Total RNA of WFTs was extracted with the RNAiso Plus reagent (Takara) following the manufacturer’s protocol. After digestion with a gDNA digester (Yeasen), cDNA was synthesized using the RevertAid First-Strand cDNA Synthesis Kit (Yeasen). The reaction mixture for PCR amplification consisted of 2 μL of cDNA, 0.25 μL of forward and reverse primers, and 5 μL TB Green Premix Ex Taq II (Takara) in a final 10-μL reaction volume. The relative expression levels of target genes were calculated by the 2-ΔΔCT method (54 (link), 55 (link)). Gene expression levels in WFTs were measured by qRT-PCR between day 2 and day 5 of the bioassay. The elongation factor 1α (EF1α) was chosen as a reference gene (56 (link)).
7
Macrophage Cultivation and Gene Expression
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The procedure for macrophage cultivation was the same as that mentioned above. The cell inoculation density was 2 × 105 cells/well, and 50 μM was set as the final concentration in the formal experiment by screening in the pre-experiment. Trizol reagent (YEASEN, Shanghai, China) was used to extract the total RNA, and Hifair® III 1st Strand cDNA Synthesis SuperMix for quantitative polymerase chain reaction (qPCR) (gDNA Digester+) (YEASEN, Shanghai, China) was used to synthesize cDNA for the Roche LightCycler® 96 RT-qPCR platform’s real-time polymerase chain reaction (PCR) (Roche, Basel, Switzerland). The primer sequences used for qRT-PCR are listed in Table 1 , with mouse TNF-α, IL-1β, and IL-6 as the specific primers and β-actin mRNA as an internal reference.
8
Jejunal Total RNA Extraction and cDNA Synthesis
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Total RNA from jejunal tissue samples was obtained using Trizol reagent (TaKaRa Biotechnology Co. Ltd., Dalian, China). The purity and quantity of the total RNA were assessed with a spectrophotometer (Pultton P200CM, San Jose, CA, United States). Subsequently, the DNA of total RNA was removed by incubation for 2 min at 42°C with a gDNA digester (Yeasen Biotechnology Co., Ltd. Shanghai, China). Total RNA was reverse transcribed to cDNA on Labcycler (SensoQuest GmbH, Göttingen, Germany) using Hifair® II SuperMix plus (Yeasen Biotechnology Co., Ltd. Shanghai, China). The reactions were incubated for 5 min at 85°C, 30 min at 42°C, and 5 min at 85°C.
9
Total RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated using Total RNA Isolation Reagent (Biosharp Life sciences, Beijing, China) according to the manufacturer’s instructions. Reverse transcription was performed using Hifair II 1st Strand cDNA Synthesis SuperMix with a gDNA digester (Yeasen, HB181210, Shanghai, China). Quantitative real-time PCR was performed using SYBR Green Master Mix (Yeasen, HB181203, Shanghai, China) on a Lightcycler Real-Time PCR System (Roche, Beijing, China). The relative expression level of the target genes and the relative fold change were normalized to GAPDH and the control, respectively. The sequences of primers (Tianyihuiyuan, Guangzhou, China) are shown in Table S2 .
10
RNA Extraction and qRT-PCR Analysis
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RD cells were harvested for RNA extraction using the AxyPrepTM Body Fluid Viral DNA/RNA Miniprep Kit (Cat No. AP-MN-BF-VNA-250, Hangzhou, Zhejiang, China) and the AxyPrepTM Multisource Total RNA Miniprep Kit (Axygene, Cat No. AP-MN-MS-RNA-250G) according to the manufacturer’s instructions. Reverse transcription was performed using a Hifair II 1st Strand cDNA Synthesis Kit with a gDNA digester (Yeasen Biotech, Cat No.11121ES60, Shanghai, China). A qRT-PCR was performed using the QuantStudio 1 Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with Hieff qPCR SYBR Green Master Mix (Yeasen Biotech, Cat:11202ES08, Shanghai, China). The primer sequences used for qRT-PCR are listed in Table 2 .
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