Osteogenic Gene Expression Analysis in rBMMSCs

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).

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Zhang H., Komasa S., Mashimo C., Sekino T, & Okazaki J. (2017). Effect of ultraviolet treatment on bacterial attachment and osteogenic activity to alkali-treated titanium with nanonetwork structures. International Journal of Nanomedicine, 12, 4633-4646.

Publication 2017

TaqMan reverse transcriptase polymerase chain reaction RNeasy Mini Kit PrimeScript RT Reagent Kit StepOne™ Plus RT-PCR System

Assay Bone morphogenetic protein Cdna Gapdh Gene expression Genes Glyceraldehyde 3 phosphate dehydrogenase Housekeeping gene Mrna Osteogenesis Osteopontin Real time polymerase chain reaction Rt pcr Runx2 Transcriptase

Corresponding Organization :

Other organizations : Osaka Dental University, Stomatology Hospital, Southern Medical University, Osaka University

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Variable analysis

independent variables

Culture duration (3, 7, 14, or 21 days)

dependent variables

MRNA levels of osteogenesis-related genes encoding ALP, Runx2, BMP, and OPN

control variables

Reverse transcriptase polymerase chain reaction (RT-PCR) assay
RBMMSCs cultured on sample discs
Total RNA isolation using RNeasy Mini Kit
CDNA synthesis using PrimeScript RT Reagent Kit
Quantification using StepOne™ Plus RT-PCR System
Normalization to the housekeeping gene GAPDH

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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