Strong-positive samples from PCV2-positive samples were selected by qPCR, and the standard curve was established. The qPCR amplification of PCV2 was carried out in 20-μL reaction mixtures containing 10 μL of TB Green II (TaKaRa), 0.8 μL of Forward primer (F), 0.8 μL of Reverse primer (R), 0.4 μL of ROX and 50 ng of DNA template. A strong linear relationship was also evident, with an R2 of 0.999, and the E was 100.91%. The linear relationship between Ct and the copy number was calculated as: Ct = − 3.3X+ 31.35. Based on geographic region, prevalence time and positive rate, representative strains were selected from positive samples for genome sequencing. The selected positive samples used primers F2/R2 to amplify the genome. Subsequently, positive PCR products 1767 nts in length were purified using the DNA Glue recovery kit (TaKaRa). The purified PCV2 genomic DNA products were ligated into the PMD19-T vector (TaKaRa) and then used to transform Escherichia coli DH5α competent cells (TaKaRa). Positive bacterial suspensions were sent to Shanghai Huada Corporation for sequencing. Whole genome was sequenced twice, and the sequence was submitted to the NCBI GenBank database.
Tb green 2
Manufactured by Takara Bio
Sourced in Japan
TB Green II is a high-performance real-time PCR reagent used for DNA detection and quantification. It is a fluorescent dye that binds to double-stranded DNA, allowing for sensitive and accurate measurement of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Rox dye 2, by Takara Bio (2 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Escherichia coli dh5α competent cells, by Takara Bio (1 mentions)
Real time pcr detection system, by Bio-Rad (1 mentions)
Trizol reagent, by Mei5 Biotechnology (1 mentions)
Mirna first strand cdna synthesis tailing reaction, by Sangon (1 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Perfect real time, by Takara Bio (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
M mlv, by Promega (1 mentions)
Trizol reagent, by Aidlab (1 mentions)
Thermal cycler dice real time system, by Takara Bio (1 mentions)
12 protocols using tb green 2
1
Genomic Sequencing of PCV2 Isolates
2
Reverse Transcription and qPCR Analysis
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Total RNA was isolated from cells using TRIzol® reagent (Mei5 Biotechnology Co., Ltd.) and then reverse transcribed (PrimeScript RT reagent kit, Takara Biotechnology, Co., Ltd.) into cDNA (reaction conditions: 42°C for 2 min followed by hold at 4°C; 37°C for 15 min, 85°C for 5 sec, and holding at 4°C). For the detection of miR-382, miRNA was reverse transcribed (miRNA First-Strand cDNA Synthesis, Tailing Reaction, Sangon Biotech Co., Ltd.) into cDNA with the polyA tailing protocol (reaction conditions: 37°C for 60 min, 85°C for 5 min, then holding at 4°C). Subsequently, 1 µl cDNA was mixed with a 9 µl PCR assay mixture containing 0.4 µM of each primer and 5 µl TB Green II (Takara Biotechnology, Co., Ltd.). PCR was conducted using the Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). β-actin was used as the internal reference for tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-10 and transforming growth factor-β (TGF-β) expression, and U6 (sequence not available) was used as the internal reference for miR-382 expression. The specific primer sequences used are presented in Table SI . The reaction conditions were 96°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 55°C/60°C for 30 sec. After the reaction, the experimental results were analyzed using the 2−ΔΔCq method (25 (link)).
3
Quantitative Real-time PCR Analysis of D. officinale
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The total RNA from the roots and leaves of D. officinale was extracted using the TRIZOL method according to the instructions of the PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa). Reverse transcription was completed to obtain cDNA samples, which were stored in a −20 °C freezer for future use. The obtained cDNA sample was diluted three times for quantitative real-time PCR (qRT-PCR) experiments (Rotor-gene Q, Kaijie Enterprise Management (Shanghai) Co., Ltd., Shanghai, China). During the qRT-PCR operation, samples were added to a 72-well plate, and each sample was subjected to 3 biological replicates. The reaction system (10 μL) was as follows: TBGREEN II (TaKaRa) 5 μL, 0.4 upstream and 0.4 downstream primers each μL, cDNA 1 μL, and ddH2O 3.2 μL. Using the Rotor-Gene Q Series software 2.0.2, the reaction procedure was carried out as follows: 95 °C for 1 min, 95 °C for 20 s, 60 °C for 30 s, 72 °C for 20 s, and finally denature at 95 °C for 1 min, anneal to 60 °C, hold for 1 min, and then rise to 95 °C at a rate of 1 °C/s to draw a dissolution curve. Three replicates were obtained for each sample. The β-actin gene is used as an internal reference, and the primer sequence is shown in Table 1 .
4
Quantifying mRNA in Mouse Hearts
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The quality and quantity of total mRNA isolated from mouse hearts using TRIzol were assessed using the Nanodrop 2000 (Thermo Fisher Scientific, USA). Samples with 260/280 and 260/230 ratios between 1.8 and 2.0 were reverse-transcribed into cDNA and used for subsequent experiment. TB Green II (Takara, Japan) was used to quantify the PCR-amplified products, and expression levels were calculated using the ΔΔCt method.
5
Isolation and Quantification of Cardiac mRNA
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Total mRNA was isolated from mouse hearts using TRIzol according to the manufacturer’s instructions (Invitrogen). The quality and quantity of isolated RNA were assessed using Nanodrop 2000 (Thermo Fisher Scientific). Samples with 260/280 and 260/230 ratios between 1.8 and 2.0 were reverse‐transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Japan). TB Green II (Takara) was used to quantify the polymerase chain reaction‐amplified products. The expression levels were calculated using the ΔΔCt method. The primer pairs used are listed in Table S1 .
6
Quantifying Gene Expression in Gastric Antrum
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Total RNA in the gastric antrum tissue was extracted and reversely transcribed into cDNA (reaction conditions: Step 1: 37℃, 15 min; Step 2: 85℃, 5 s; Step 3: 4℃). 10 ul total reaction system consisting of cDNA template, TB Green II (Cat NO.: RR820A, Takara Bio Inc., Japan), Rox, primers (Sangon Biotech, Shanghai, China) (Table 3 ), and RNase-free water were prepared for RT-qPCR amplification (amplification conditions: stage 1: 95℃, 30 s, 1 cycle; stage 2: 95℃, 5 s, 60℃, 34 s, 40 cycles). 2−ΔΔCt represented relative mRNA expression and was used for statistical analysis.
Primers for RT-qPCR test
| Gene | sequence | length (bp) |
|---|---|---|
| Arg-1 | Forward: cag tat tca ccc cgg cta | 150 |
| Reverse: cct ctg gtg tct tcc caa | ||
| iNOS | Forward: cgg aga aca gca gag ttg g | 149 |
| Reverse: gga ata gca cct ggg gtt t | ||
| NF-κB(p65) | Forward: ttc ctg ggg aga gaa gca | 114 |
| Reverse: ggt gag gtg ggt ctt tgg | ||
| IκBα | Forward: cat ctc cac tcc gtc ctg | 110 |
| Reverse: gca ccc aaa gtc acc aag | ||
| β-Actin | Forward: cct cac tgt cca. cct tcc a | 120 |
| Reverse: ggg tgt aaa acg cag ctc a |
7
RT-qPCR for Gene Expression Analysis
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RNA from the cultured cells was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted RNA using the iScript cDNA synthesis kit (Bio-Rad). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using 4 ng/μL cDNA, TB Green II, and Rox Dye II (TaKaRa) with 20 μM of each primer. The amplification was carried out using a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific 4453723) according to the manufacturer's instructions. Primers are summarized in Supplementary Table 2 .
8
RNA Extraction and RT-qPCR Analysis
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RNA from the cultured cells was extracted using the RNeasy Mini Kit (Qiagen). Subsequently, cDNA was synthesized from the extracted RNA using the iScript cDNA synthesis kit (Bio-Rad). RT-qPCR was conducted utilizing 4 ng/μL cDNA, TB Green II, and Rox Dye II (TaKaRa), along with 20 μM of each primer. The amplification was carried out using a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific 4453723) in accordance with the manufacturer's instructions. The detail of the primers used for qPCR was summarized in Supplementary Table 2 .
9
Quantitative Real-Time PCR Analysis
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Total cDNA was synthesized from 1,000 ng RNA according to the manufacturer’s instructions (Perfect Real Time; TaKaRa Biotechnology, Dalian, China). qRT-PCR Primers are listed in Table S3
10
Quantitative PCR of Ovarian Gene Expression
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Total RNA from the ovary was isolated and reverse transcribed into cDNA according to the techniques described in the previous work.29 (link) Primer Express Software (Applied Biosystems, Foster City, CA, USA) was used to design the primers, which were then manufactured by TSINGKE Biological Technology (Beijing, China). Table S1
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