Cells were harvested after 1, 2, and 4 hours of the application of compressive force. Total RNA was extracted using an EASYspin RNA extraction reagent kit (Biomed, Beijing, China) from cells cultured on the plates according to the manufacturer's instructions. RNA was reverse-transcribed using the PrimerScript® RT reagent kit (Takara, Dalian, China) with gDNA eraser to synthesize cDNA. qPCR (n = 3) was performed using SYBR Green Premix Ex Taq (Takara) and the MxPro Mx3005P real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalization control. The primers used for qPCR were shown in Table 1 . The cycling conditions were as follows: 95℃ for 30 seconds followed by 40 cycles of 95℃ for 5 seconds, 55℃ for 30 seconds, and 72℃ for 1 minute. The 2-ΔΔCt method was used for calculating the relative expression levels, and values were the average of triplicate measurements.
Mxpro mx3005p real time pcr detection system
Manufactured by Agilent Technologies
Sourced in United States
The MxPro Mx3005P is a real-time PCR detection system manufactured by Agilent Technologies. It is designed for the detection and quantification of nucleic acid sequences. The system utilizes fluorescence-based detection methods to monitor the amplification of target DNA or RNA in real-time during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using mxpro mx3005p real time pcr detection system
1
Compressive Force-Induced Gene Expression
2
Mechanical Force-induced miRNA and mRNA Expression
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The total RNAs from rBMSCs (after 7 days) and alveolar bone tissue (after 14 days) after mechanical force loading were isolated using a HiPure Total RNA kit according to the manufacturer's instructions. MiRNA was subjected to reverse transcription and PCR by using an SYBR Green Hairpin-it miRNAs qRT-PCR kit. The mRNAs were investigated with a PrimerScript® RT reagent kit and SYBR Green Premix Ex Taq. The PCR products were evaluated with a MxPro Mx3005P real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA). The internal controls of mRNAs and miR-34a were β-actin and U6, respectively. The cycling conditions of mRNAs were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 55°C for 30 s, and 72°C for 1 min. The cycling conditions for miRNAs were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 12 s and 62°C for 40 s. The 2-ΔΔCt method was used to calculate the relative expression levels, and the obtained values were averaged from triplicate measurements.
3
Osteoclastogenesis Assay in RAW264.7 Cells
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RAW264.7 cells were induced to form osteoclasts using mouse RANKL (50 ng/mL; Peprotech, Rocky Hill, NJ, USA) for 4 days. Total RNA was isolated using a HiPure Total RNA kit (Megan, Guangzhou, China) according to the manufacturer’s instructions. The mRNAs were investigated with a PrimerScript® RT reagent kit and SYBR Green Premix Ex Taq. The PCR products were evaluated with a MxPro Mx3005P real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA). The internal control mRNA was β-actin. The cycling conditions of mRNAs were: 95°C for 30s, followed by 40 cycles at 95°C for 5s, 55°C for 30s, and 72°C for 1 min. The 2-ΔΔCt method was used to calculate the relative expression levels, and the obtained values were averaged from triplicate measurements.
4
Osteoclast Differentiation Kinetics
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RAW264.7 cells were induced to differentiate into osteoclasts by treatment with mouse Receptor Activator of NF-κB Ligand (RANKL) (50 ng mL−1, Peprotech, USA) for 2, 4, and 6 days. Then, the total RNAs were isolated using a HiPure Total RNA kit according to the manufacturer's instructions. The mRNAs were investigated with a PrimerScript® RT reagent kit and SYBR Green Premix Ex Taq. The PCR products were evaluated with a MxPro Mx3005P real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA). The internal control for mRNAs was β-actin. The cycling conditions of mRNAs were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 1 min. The 2−ΔΔCt method was used to calculate the relative expression levels, and the obtained values were averaged from triplicate measurements.
5
Quantifying Inflammatory Cytokine Expression
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Liquid nitrogen was used to freeze the gingival tissue sample, which was then crushed. Total RNA was isolated using a HiPure Total RNA kit (Yeason, China) according to the manufacturer's instructions. The mRNAs were was subjected to reverse transcription using Hifair II 1st Strand cDNA Synthesis SuperMix for qPCR (Yeason, China) and qPCR using Hifair qPCR SYBR GREEN Master Mix (Yeason, China). The PCR products were evaluated with a MxPro Mx3005 P real-time PCR detection system (Agilent Technologies, Santa Clara, CA, USA). The internal control for mRNAs was β-actin. The cycling conditions of mRNAs were as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min. The 2−ΔΔCt method was used to calculate the relative expression levels, and the obtained values were averaged from triplicate measurements. The primers were as follows:
TNF-α(F):5′-ACCCTCACACTCACAAACCAC3'/
(R):5′-ACAAGGTACAACCCATCGGC-3';
IL-10(F):5′-GCATGGCCCAGAAATCAAGG-3'/
(R):5′-ACACCTTGGTCTTGGAGCTTATTA-3';
IL-1(F):5′-GCCACCTTTTGACAGTGATGAG-3'/
(R): R:5′-AGCTTCTCCACAGCCACAAT-3'.
TNF-α(F):5′-ACCCTCACACTCACAAACCAC3'/
(R):5′-ACAAGGTACAACCCATCGGC-3';
IL-10(F):5′-GCATGGCCCAGAAATCAAGG-3'/
(R):5′-ACACCTTGGTCTTGGAGCTTATTA-3';
IL-1(F):5′-GCCACCTTTTGACAGTGATGAG-3'/
(R): R:5′-AGCTTCTCCACAGCCACAAT-3'.
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