1 × 105 cells/well were seeded in 6-well plates. Total RNA was extracted by the use of a RNA Extraction Kit (TAKARA, Japan). The concentration and purity of extracted RNA were evaluated by NanoDrop (Thermo Scientific, USA). After cDNA was synthesized, real-time quantitative PCR was conducted using SYBR® Premix Ex Taq™ II (TAKARA, Japan) on an ABI QuantStudio 3 PCR System (Applied Biosystems, USA). The primer sequences for HMGB1, RAGE, heme oxygenase-1, Runx2, ALP, OCN, OPG, RANKL, and β-actin are listed in Table 1 . The expressions of detected genes were figured out by 2−∆∆CT method.
Abi quantstudio 3 pcr system
Manufactured by Thermo Fisher Scientific
Sourced in China
The ABI QuantStudio 3 PCR System is a real-time PCR instrument designed for quantitative analysis of nucleic acids. It is capable of performing fast, precise, and reliable qPCR experiments with a compact footprint.
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Lab products found in correlation
4 protocols using abi quantstudio 3 pcr system
1
Gene Expression Analysis of Osteoblastogenesis
2
Cellular and Zebrafish RNA Extraction
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For cellular RNA extraction, 400 µL TRIzol was added to the cell culture wells for lysis. For zebrafish RNA extraction, 10 zebrafish were placed into 1.5-mL EP tubes, and 400 µL TRIzol added for lysis (38 (link)). RNA was isolated and purified according to the manufacturer’s instructions for the use of TRIzol reagent (TaKaRa, Japan). RNA was reverse transcribed into cDNA using a cDNA Reverse Transcription Kit (Monad, Shanghai, China). Real-time quantitative PCR (RT-qPCR) analysis was performed using SYBR Green PCR Mix (Monad, Shanghai, China) in the ABI QuantStudio 3 PCR system (Applied Biosystems, Waltham, MA, USA). The primers used are listed in Table 1 .
3
Quantitative RT-PCR Analysis of Osteogenic Genes
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Total RNA was extracted using the RNA Extraction Kit (TAKARA, Japan) following the manufacturer's instruction and the RNA quality confirmed by NanoDrop (Thermo Scientific). After cDNA was synthesized with the PrimeScriptRT reagent Kit (TAKARA), the qRT‐PCR assay was performed using SYBR Premix Ex Taq II (TAKARA) on an ABI QuantStudio 3 PCR System (Applied Biosystems, USA). Relative gene quantitation was determined by the 2‐ΔΔCT method. The primer sequences for COL1α1, ALP, OPG, Runx2, OCN, Drp 1, SOD1, and GAPDH are listed in Table S1 (Supporting Information).
4
Quantification of Fungal Gene Expression
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The RNA extraction and construction of cDNA libraries was performed as described previously (He et al., 2021 (link); Wei et al., 2022 (link)). Conidia of F. oxysporum (1 × 106 CFU) were added to PDB medium and incubated for 24 h. The mycelia were collected and ground to a powder in liquid nitrogen. Total RNA was extracted from the ground material using RNAiso Plus (TaKaRa, Japan). Real-time, quantitative PCR (RT-qPCR) analysis was performed with a SYBR Green master mix (Monad, Shanghai, China) and the ABI QuantStudio 3 PCR system (Applied Biosystems, Waltham, MA, USA). Relative expression levels of the genes were calculated using the threshold cycle (2−ΔΔCT also known as 22DDCT) method (Livak and Schmittgen, 2001 (link)). Gene expression levels were normalized against the expression of the 18S rRNA housekeeping gene (Table 3 ). Details regarding the relevant primers are provided in Supplementary Table S3 .
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