The expression of osteogenic marker genes, RUNX2, collagen I (Col I), and OPN, was analyzed by real-time polymerase chain reaction (RT-PCR). The total RNA concentration and purity were determined by a Nanodrop Assay (Tecan M200). The first strand cDNA was synthesized by reverse transcriptase as described in the M-MLV manual (Promega). Gene-specific primers were designed using the primer design software Beacon 5.0. The specificity of oligonucleotides was checked by BLASTN® (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI. All samples were assayed in triplicate in 8 striped optical tubes (Axygen) using a qPCR SYBR Green Mix Kit (Stratagene). The PCR amplification was performed as follows: initial heating at 95°C for 10 min, followed by 40 cycles at 95°C for 30 s, 58°C for 60 s, and 72°C for 60 s. Each gene expression value was normalized to that of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results are reported as the relative gene expression. Information on the primers is provided in Table 1 .
M mlv manual
Manufactured by Promega
The M-MLV manual is a reverse transcriptase enzyme used for the conversion of RNA to cDNA. It is a key component in the process of gene expression analysis and is often utilized in various molecular biology applications.
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5 protocols using m mlv manual
1
Osteogenic Marker Gene Expression Analysis
2
Nanofibrous Platform for NSC Differentiation
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NSC differentiation on electrospun nanofibres was assessed by gene expression using real-time polymerase chain reaction (RT-PCR) on day 7. The total RNA concentration and purity were detected by a Nanodrop system (Tecan M200). First-strand cDNA was synthesized by reverse transcriptase as described in the M-MLV manual (Promega). Gene-specific primers were designed using Beacon 5.0 primer design software. The specificity of oligonucleotides was verified by BLASTN® (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI. All samples were analysed in triplicate in 8-striped optical tubes (Axygen) using a qPCR SYBR Green Mix Kit (Stratagene). PCR amplification was performed as follows: initial heating at 95 °C for 10 min, followed by 40 cycles at 95 °C for 30 s, 58 °C for 60 s, and 72 °C for 60 s. Each gene expression value was normalized to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results were reported as the relative gene expression. The genes and primer information is shown in Table 1 .
3
Quantitative Real-Time PCR Analysis
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The mRNA of cells was extracted using trizol (Invitrogen) and collected using the Qiagen RNEasy Extraction kit (Qiagen). Samples were stored at -80°C until reversed transcription. Total RNA concentration and purity were detected by Nanodrop Assay (Tecan M200). The first strand cDNA was synthesized by reverse transcriptase as described of M-MLV manual (Promega). Gene-specific primers including GAPDH, COL I, COL II, OPN, and Runx2 were designed using the primer design software of beacon 5.0 (Table 1 ). All samples were performed in triplicates in 8 striped optical tube (Axygen) using qPCR SYBR Green Mix Kit (Stratagene) The amplification efficiencies of primers was verified by cDNA serial 5 times dilutions. The qPCR amplification was done as follows: initial heating at 95°C for 10min, followed by 40 cycles at 95°C for 30 s, 58°C for 60s, 72°C for 60 s. Specificity of listed oligonucleotides were checked by BLASTN® (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI.
4
Quantitative Analysis of Osteogenic Markers
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The MC3T3-E1 cells cultured on various nanofibrous matrices for 7 days were also collected for the evaluation of the osteogenesis-related genes expression. Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. The total RNA concentration and purity were detected by a Nanodrop assay (Tecan M200), and the first strand cDNA was synthesized by reverse transcriptase as described in theM-MLV manual (Promega). The expression of osteogenic markers was quantified by qPCR SYBRGreen Mix Kit (TaKaRa). The primer sequences specific for the target gene including anti-runt-related transcription factor 2(RUNX2), osteopontin (OPN) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used for qRT-PCR are listed in Table 1 . The specificities of the listed oligonucleotides were checked by BLASTN® (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI. The qPCR amplification was done as follows: initial denaturation at 95°C for 10min, followed by 40 cycles at 95°C for 30 s, 58°C for 1 min, 72°C for 1 min. The comparative threshold cycle method was used to analyse the Q-PCR results using iCycleriQ Detection System software with GAPDH as the reference gene. All results were quantified using the ΔΔCt relative quantification method.
5
Quantifying Osteogenic Gene Expression
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Total intracellular RNA was isolated from incubated cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The concentration and quality of the isolated RNA were evaluated using a nanodrop spectrophotometer (Shimaduz, Japan). One milligramme of total RNA was used to produce cDNA using reverse transcription as described by the MMLV manual (Promega). The expression of osteogenic markers was quantified by a qPCR SYBRGreen Mix Kit (Takara). Target gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The specific primer sequences for the target gene used for qRT-PCR, including those for the anti-runt-related transcription factor 2 (RUNX2), osteopontin (OPN) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are listed in Table 1 .
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