Total RNA was extracted by Trizol reagent (Invitrogen - Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. cDNA was produced using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems-Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. mRNA levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) using the TaqMan Gene Expression Assays in 7900HT Real-Time PCR System (Applied Biosystems-Thermo Fisher Scientific, Waltham, MA, USA). The CTSK (Hs00166156_m1), ACP5 (Hs00356261_m1), MMP9 (Hs00234579_m1), MMP2 (Hs001548727_m1), and CALCR (Hs001016882_m1) expression levels were normalized to the endogenous housekeeping gene Glucuronidase Beta (GUSβ) (Hs99999908_m1).
Taqman gene expression assays in 7900ht real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan Gene Expression Assays are a set of standardized, pre-designed qPCR assays for gene expression analysis. They are compatible with the 7900HT Real-Time PCR System, which is a high-throughput, automated instrument for real-time PCR applications.
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2 protocols using taqman gene expression assays in 7900ht real time pcr system
1
Quantitative Real-Time PCR Analysis of Bone Resorption Genes
2
Quantifying Osteoclast and Osteoblast Gene Expression
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Total RNA was extracted from osteoclast and osteoblast cells at the end of the differentiation protocol using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA was treated with DNase buffer and DNase (DNAse Turbo, Applied Biosystems) to avoid genomic DNA contamination. cDNA was produced using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer's instructions. mRNA levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) using TaqMan Gene Expression Assays in 7900HT Real-Time PCR System (Applied Biosystems). Tartrate resistant acid phosphatase (TRAP) (Hs00356261_m1), cathepsin-K (Hs00166156_m1), metalloproteinase-9 (Hs00234579_m1), alkaline phosphatase (ALP) (Hs01029144_m1), osteocalcin (Hs00234160_m1) and runt-related transcription factor 2 (RUNX2) (Hs00231692_m1) expression levels were normalized to the endogenous housekeeping gene glucuronidase beta (GUSb) (Hs99999908_m1) in both untreated and treated samples using the ΔCT calculation. Subsequently relative expression levels in treated samples were normalized to the mRNA levels detected in control samples using the ΔΔCT calculation [21 ].
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