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.
Electric homogenizer
Manufactured by IKA Group
The Electric Homogenizer is a laboratory equipment used for homogenizing, dispersing, and mixing a variety of samples. It features a powerful electric motor that drives a high-speed rotor, which creates a strong shearing and mixing action to thoroughly blend the sample components.
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2 protocols using electric homogenizer
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.
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