PureZOL™ RNA Isolation Reagent

Protocol tips

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- This kit can be used for the isolation of RNA from up to 5mg tissue

Upstream tips
- This kit can be used for the isolation of RNA from up to 5mg tissue

Publication protocol

Tibiae were dissected from the rats and cleaned with 0.1 M ice-cold PBS, pH 7.2. For OVX+SW and OVX+RAL+SW groups, both contralateral and SW-treated tibiae were used. The epiphysis of each bone was excised and the bone marrow was collected [28]. Bone tissues were snap-frozen in liquid nitrogen to make them breakable. Total RNA was extracted using TRIzol Reagent (Bio-Rad Laboratories), according to the manufacturer’s instructions. cDNA was synthesized using a reverse transcription kit (NucleoSpin®, MACHEREY-NAGEL GmbH & Co, Düren, Germany) from 2 μg total RNA. PCRs were performed with a Bio-Rad CFX96 Connect Real-time PCR System instrument and software (Bio-Rad Laboratories). The PCR conditions were 15 min at 95°C followed by 40 cycles of two-step PCR denaturation at 94°C for 15 s, annealing at 55°C for 30 s and extension at 72°C for 30 s. Each sample contained 1–100 ng cDNA in 2X QuantiTect SYBRGreen PCR Master Mix and primers, TNF-α, RANKL, OPG, Bone morphogenetic 2 (Bmp2), runt-related transcription factor 2 (RUNX2), cathepsin k, Peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin (both by Qiagen, Hilden, Germany) in a final volume of 50 μl. The relative amount of each studied mRNA was normalized to GAPDH as housekeeping gene, and data were analyzed according to the 2-ΔΔCT method.

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Manufacturer protocol

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