Quantitative PCR was performed on SH-SY5Y cell samples and on spinal cord samples obtained from mice. Total RNA was isolated by a standard method with TRIZOL reagent (Invitrogen Life Technologies). Isolated RNA was reverse-transcribed into cDNA using PrimeScript™ RT Reagent Kit (TaKaRa) following standard protocols. Real-time quantitative PCR (qPCR) was performed with synthetic primers and SYBR Green (TaKaRa) with a Quant-Studio 5 Real-Time PCR Detection System (Thermo Fisher Scientific). The relative expression level of SQSTM1/p62 was calculated and quantified with the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)) after normalization with the reference GAPDH and ACTB expression. All primers used are listed in Table 1 .
Quantstudio 5 real time pcr detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Japan
The QuantStudio 5 Real-Time PCR Detection System is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of performing precise and sensitive DNA and RNA detection and quantification across a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Sybr green, by Takara Bio (3 mentions)
Hiscript 2 q rt supermix for qpcr, by Vazyme (3 mentions)
Chamq sybr qpcr master mix, by Vazyme (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Fast start 4x probe master mix, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Magmax viral pathogen 2 nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Power up sybr green kit, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Magmax viral pathogen nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr machine, by Bio-Rad (1 mentions)
Kingfisher flex purification system, by Thermo Fisher Scientific (1 mentions)
20 protocols using quantstudio 5 real time pcr detection system
1
Quantitative PCR Analysis of SQSTM1/p62 Expression
2
SARS-CoV-2 RT-qPCR Quantification Protocol
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Viral RNA was automatically extracted from 200 μL of the NPS specimens using the MagMAXTM Viral/Pathogen II Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Vilnius, Lithuania) on KingFisher (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RT-qPCR was performed using TaqPath™ COVID-19 multiplex real-time RT-PCR test (the Orf1ab, N and S genes) (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Negative and positive controls were run simultaneously with samples [2 (link)]. The assay was performed using a QuantStudio 5 real-time PCR detection system (Thermo Fisher Scientific, Waltham, MA, USA). Test results were reported quantitatively as cycle threshold (Ct) value of the ORF1ab, the N and the S genes, while qualitative data were reported as positive/negative at test cut-off (i.e., Ct < 37 for positive or ≥ 37 for negative samples). The Ct values used to classify specimens as ‘high viral load’, ‘medium viral load’ and ‘low viral load’ defined as previously described [7 (link),21 (link)], they were <18.57, 18.57–28.67 and >28.67, respectively.
3
Quantitative PCR for M. genitalium
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Commercially available primers and probes specific for M. genitalium (Ba04646249_sl) were used with the TaqMan quantitative polymerase chain reaction (qPCR) assay (ThermoFisher Scientific, USA). The assay was run on the Quant Studio 5 Real-time PCR detection system (ThermoFisher Scientific, USA). The PCR was performed in a final reaction volume of 5 µL comprising: 0.25 µL Fluorescein amidite (FAM)-labelled probe and/or primer mix, 1.25 µL Fast Start 4x probe master mix (ThermoFisher, Part No. 4444434), 1.5 µL template DNA and nuclease-free water. Non-template control reactions and positive controls (TaqMan™ Vaginal Microbiota Extraction Control; cat no. A32039, ThermoFisher Scientific, USA) were also included. Amplification was performed at 95 °C for 30 s followed by 45 cycles comprising of denaturation at 95 °C for 3 s and annealing at 60 °C for 30 s. Detection of amplified fluorescent products was carried out at the end of the annealing phase. The raw fluorescent data included the CT mean values, which were automatically generated by the Quant Studio 5 Real-time PCR system software (ThermoFisher Scientific, USA).
4
Trichomonas vaginalis Detection Protocol
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A total of n = 362 vaginal swab DNA samples were tested for the presence of T. vaginalis. T. vaginalis was detected using the Applied Biosystems™ TaqMan® Assays. Commercial primers and probes (Pr04646256_s1) which target the alpha tubulin 1 gene of T. vaginalis were used. Amplification was performed on the QuantStudio 5 Real-Time PCR Detection System (ThermoFisher Scientific, USA). Briefly, each reaction was performed in a final volume of 5 μl that comprised 0.5 μl FAM-labelled probe/primer mix for individual targets, 2.5 μl FastStart 4x probe master mix (ThermoFisher Scientific, Pr04646256_s1), 1.5 μL template DNA, and nuclease-free water. We also included nontemplate control reactions. Amplification was performed under the following conditions: 1 cycle at 95°C for 30 seconds followed by 45 cycles of denaturation at 95°C for 3 seconds and annealing at 60°C for 30 seconds. The detection of fluorescent products was performed at the end of the annealing period. The raw fluorescent data that included the CT mean values were automatically generated by the QuantStudio 5 Real-Time PCR System software.
5
Quantifying XIST Expression in Breast Cancer
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Total RNA was extracted from patients’ breast cancer at the First Affiliated Hospital of Nanjing Medical University using TRIzol reagent (Invitrogen, CA, USA). Isolated RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (Vazyme Biotech Co.,Ltd. Nanjing, China) following standard protocols. Real-time quantitative PCR (qPCR) was performed with synthetic primers and ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China) with a Quant Studio 5 Real-Time PCR Detection System (Thermo Fisher Scientific, MA, USA). The relative expression levels of XIST were calculated and quantified with the 2−ΔΔCt method after normalization with the reference β-actin expression. All primers used are listed in Table 1
6
Quantitative Real-Time PCR Analysis of Inflammatory Genes
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Total RNA was extracted from BV-2 cells using Trizol reagent (Invitrogen, CA, USA). Isolated RNA was reverse transcribed into cDNA using Prime-Script™ RT Reagent Kit (TaKaRa, Japan) following standard protocol. Quantitative real-time PCR (qPCR) was performed with synthetic primers and SYBR Green (TaKaRa, Japan) with QuantStudio 5 Real-Time PCR Detection System (Thermo Fisher scientific). The relative expression levels of Il1b and Tnfa were calculated and quantified with the 2−ΔΔCt method after normalization with reference. All primers used are listed in Table 1 .
Sequences of primers for real-time quantitative polymerase chain reaction
| Gene | Sequence | |
|---|---|---|
| Gapdh | Forward | 5′-GGCATGGACTGTGGTCATGAG-3′ |
| Reverse | 5′-TGCACCACCAACTGCTTAGC-3′ | |
| Illb | Forward | 5′-TCATTGTGGCTGTGGAGAAG-3’ |
| Reverse | 5′-AGGCCACAGGTATTTTGTCG-3’ | |
| Tnfa | Forward | 5′-CATCTTCTCAAAATTCGAGTGACAA-3’ |
| Reverse | 5′-TGGGAGTAGACAAGGTACAACCC-3’ |
Gapdh glyceraldehyde 3-phoshate dehydrogenase, Illb interleukin-1β, Tnfa tumor necrosis factor-α
7
Strand-specific qPCR to Detect Pig Diarrhea
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In 2022, small intestinal tissues were collected from a diarrhoeal pig farm in Heilongjiang Province, China, with clinical symptoms of watery diarrhoea, vomiting, dehydration and rapid weight loss. Intestinal contents of infected piglets from the same house were mixed and sample nucleic acids were extracted using Trizol™ (Thermo, USA) following the manufacturer’s instructions. The gDNA Eraser-treated RNA samples were reverse-transcribed with strand-spe-cific RT primers at 42 °C for 15 min with the PrimeScript® Reverse Transcriptase (Takara, China). Strand-specific quantitative PCR (qPCR) was performed with gene-specific primers and the LightCycler® 480 SYBR Green I Master (Roche, Switzerland) on the QuantStudio™ 5 Real-Time PCR Detection System (Thermo, USA). ORF3 plasmid, flat used as an internal control to normalize gene expression, was kept by the laboratory.
8
SARS-CoV-2 Viral RNA Quantification in Respiratory Samples
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RNA was extracted from the lung tissues (mice and hamsters) and nasal washes (hamsters) using the TRIzol LS Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The concentration and purity of the extracted RNAs were determined using NanoDrop. To quantify SARS-CoV-2 viral RNA copies, RT-PCR was performed using the PowerUP SYBR Green Kit (Thermo Fisher) and the QuantStudio 5 Real-Time PCR Detection System (Thermo Fisher). The Throat Swab sample was analyzed for SARS-CoV-2-specific RNA by quantitative RT-PCR (qRT-PCR). As recommended by the Centers for Disease Control and Prevention (CDC), we used ORF1ab-specific primers (forward: 5’-CCCTG TGGGTTTTACACTTAA-3’ and reverse: 5’-ACGATTGTGCATCAGCTGA-3’) to detect the viral RNA level. PCR reactions (10 μl) contained primers (10 μM), cDNA sample (1.5 μl), SYBR Green reaction mix (5 μl), and molecular grade water (2.5 μl). PCR cycling conditions were as follows: 95°C for 3 min, 45 cycles of 95°C for 5 s, and 60°C for 30 s. For each RT-PCR, a standard curve was included using an RNA standard (Armored RNA Quant®) to quantify the absolute copies of viral RNA in the throat swabs.
9
Quantitative RT-PCR Gene Expression Analysis
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Total mRNA was extracted and purified from 3 individual samples per condition using RNA isolation kit (NucleoSpin® RNA). We used a maximum amount of 3 μg/60 μL of mRNA for reverse transcription with PrimeScript™ RT Master Mix kit as per the manufacturer’s instructions. Template cDNA samples were diluted (1:10) in TaqMan® Universal PCR Master Mix and TaqMan® Gene Expression Assay forward and reverse primers per gene of interest and distributed into 5 wells of a 384 multi-well reaction plate. Quantitative RT-PCR was performed using the QuantStudio® 5 Real-Time PCR detection system (ThermoFisher Scientific). β-actin was used as the endogenous gene. Primer data are listed in Supplementary Table 5 .
10
RNA Extraction and qPCR Analysis
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Total RNA from LCM samples was extracted using the RNeasy micro kit (Catalog No. 74004, Qiagen Inc., Boston, MA, USA). RMTg tissue was resuspended in 30 μL lysis buffer and vortexed for 30 s, then treated following the instructions of the RNeasy micro kit for Microdissected Cryosections, and the total RNA was eluted with 10 μL of RNase-free water. RNA was quantified by a spectrophotometer Nanodrop One (Thermo scientific, Waltham, MA, USA) in absorbance ratios at 260–280 nm of 1.8–2.0. The cDNA was synthesized using the HiSlid ™ cDNA Synthesis Kit for qPCR (with dsDNase) (MIKXCo., Ltd., MKG840, Shenzhen, China). The primers were designed and obtained from Sangon Biotech. The quantitative PCR was performed on a QuantStudio™ 5 Real-Time PCR Detection System (Thermofisher, Waltham, MA, USA) using the 2 × Polarsignal@ qPCR mix (MIKXCo., Ltd., MKG800, Shenzhen, China) under the following conditions: 20 s at 94 °C followed by 45 cycles of 10 s at 94 °C and 20 s at 60 °C. Real-time PCR was performed in triplicate for each sample.
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