The expression levels of osteogenesis-related genes were evaluated using a real-time TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) assay (Life Technologies, Carlsbad, CA, USA), as previously described.7 (link) rBMMSCs were cultured on sample discs for 3, 7, 14, or 21 days. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Venlo, the Netherlands) and aliquots (10 μL) of each RNA sample were reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit (TaKaRa Bio, Shiga, Japan). The mRNA levels of the osteogenesis-related genes encoding ALP, runt-related transcription factor 2 (Runx2), bone morphogenetic protein (BMP), and osteopontin (OPN) were quantified using the StepOne™ Plus RT-PCR System (Life Technologies). ALP and Runx2 expressions were measured at 3 and 7 days, while BMP and OPN expressions were measured at 14 and 21 days. Relative gene expression levels in each group were calculated using the ΔΔCt method and were normalized to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Taqman reverse transcriptase polymerase chain reaction
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) is a laboratory technique used for the detection and quantification of specific RNA sequences. It is a combination of reverse transcription and real-time PCR, allowing for the simultaneous conversion of RNA to cDNA and the amplification and detection of the target sequence.
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2 protocols using taqman reverse transcriptase polymerase chain reaction
1
Osteogenic Gene Expression Analysis in rBMMSCs
2
Osteogenic Gene Expression Analysis
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A real-time TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) assay (Life Technologies, Carlsbad, CA, USA) was used for analyzing the expression levels of osteogenesis-related genes, as previously described [27 (link)]. After 3, 7, 14, and 21 d of culture on the samples, total RNA was extracted from rBMMSCs using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). Equal amounts (10 μL) of RNA samples were reverse transcribed into cDNA using the PrimeScript RT kit (TaKaRa Bio, Shiga, Japan). The expression levels of ALP and runt-related transcription factor 2 (Runx2) were quantitatively analyzed at 3 and 7 d, and of bone morphogenetic protein 2 (BMP-2) and bone γ-carboxyglutamate (gla) protein (Bglap) were analyzed at 14 and 21 d using StepOneTM Plus RT-PCR System (Life Technologies, Carlsbad, CA, USA). The relative gene expression in each group was normalized to that of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), using the ΔΔCt method.
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