For RNA isolation from the maxilla, soft or both soft and hard tissues (as indicated in the text) were homogenized in 1 ml TRI reagent (Sigma) using an electric homogenizer (IKA labortechnik), and RNA was extracted according to the manufacturer’s instructions. Extraction of RNA from cell cultures was executed using 200 μl TRI reagent, and RNA was extracted according to the manufacturer’s instructions. cDNA synthesis was performed using the qScript cDNA Synthesis kit (Quanta-BioSciences). Quantitative RT-PCR reactions (20 µl volume) were performed using Power SYBR Green PCR Master Mix (Quanta-BioSciences). The following reaction conditions were used: 10 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 60°C. The samples were normalized to the TBP (TATA box–binding protein) or Actin genes as control mRNA, by the change in cycling threshold (ΔCT) method, and calculated based on 2−ΔΔCT.
Power sybr green pcr master mix
Manufactured by Quanta Biosciences
Power SYBR Green PCR Master Mix is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I dye, necessary reagents, and a high-performance DNA polymerase for sensitive and reproducible gene expression analysis.
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5 protocols using power sybr green pcr master mix
1
RNA Isolation and Quantitative RT-PCR Analysis
2
Gingival and Tongue RNA Isolation and RT-PCR
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For RNA isolation, the excised gingival tissues and tongues were homogenized in 500 µl TRI reagent (Sigma) using an electric homogenizer (IKA labortechnik) and RNA was extracted according to the manufacturer’s instructions. cDNA synthesis was performed using the qScript cDNA Synthesis Kit (Quanta-BioSciences). RT-PCR reactions (10 µL volume) were performed using Power SYBR Green PCR Master Mix (Quanta-BioSciences) and specific primers to the examined gene. Sequences of the various primers used in this study are provided in the supplementary data. The following reaction conditions were used: 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 60 s at 60 °C. The samples were normalized to GAPDH as control mRNA, by change in cycling threshold (ΔCT) method and calculated based on 2-ΔΔCT.
3
RNA Isolation and qRT-PCR Analysis
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For RNA isolation from the maxilla, soft or both soft and hard tissues were homogenized in 1 mL TRI reagent (Sigma) using electric homogenizer, and RNA was extracted according to the manufacturer’s instructions. For RNA isolation from lymph nodes, tissue was homogenized manually in 0.5 mL of TRI reagent (Sigma). cDNA synthesis was performed using the qScript™ cDNA Synthesis Kit (Quanta-BioSciences Inc™). RT-qPCR reactions (20 µl volume) were performed using Power SYBR Green PCR Master Mix (Quanta-BioSciences IncTM.). The following reaction conditions were used: 10 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 60°C. The gingival samples were normalized to TBP (TATA box binding protein) gene as control and the lymph node samples to β-actin (Figure S1 in Supplementary Material). The 2−ΔΔCT method was used to calculate relative gene expression.
4
Quantitative RNA Expression Analysis
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For RNA isolation, the maxilla was homogenized in 1 ml TRI reagent (Sigma) using electric homogenizer, and RNA was extracted according to the manufacturer’s instructions. cDNA synthesis was performed using the qScript™ cDNA Synthesis Kit (Quanta-BioSciences Inc™). Real-Time qPCR reactions (20 µl volume) were performed using Power SYBR Green PCR Master Mix (Quanta-BioSciences Inc™). The following reaction conditions were used: 10 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 60°C. The samples were normalized to the TBP (TATA box binding protein) as control mRNA, by change in cycling threshold (ΔCT) method and calculated based on 2−ΔΔCT.
5
RNA Isolation and qRT-PCR Analysis
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RNA was isolated and served for cDNA synthesis using qScriptTM cDNA Synthesis Kit, (Quanta‐BioSciences Inc.). RT‐qPCR reaction was performed according to the manufacturer's instruction in a 20‐μl reaction mixture using the Power SYBR Green PCR Master Mix (Quanta‐BioSciences Inc.). For more details, see Appendix S1 .
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