Osteoblasts cells were seeded in 6-well plates at a density of 1 × 106 cells/well. After 6-day culture, cells were treated with the TPF at concentrations of 0, 50, and 100 ng/ml for 48 h. Total RNA from the cells of each well was isolated respectively using NucleoSpin (Macherey–Nagel, Duren, Germany). RNA aliquots were reverse transcribed to complementary DNAs by using an oligo (dT) primer (Roche), deoxynucleotide triphosphate (dNTP), and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas, Hanover, MD). The complementary DNA products were subjected to PCR amplification with gene-specific primers for mouse Alp, Osteocalcin and Type 1 collagen [23 (link)]. Real-time RT-PCR amplification was performed using a Light Cycler System (Roche) with a Platinum SYBR Green qPCR Super Mix UDG kit (Invitrogen, Carlsbad, CA).
Moloney murine leukemia virus m mulv reverse transcriptase
Manufactured by Thermo Fisher Scientific
Sourced in United States
Moloney murine leukemia virus (M-MuLV) reverse transcriptase is an enzyme that catalyzes the synthesis of complementary DNA (cDNA) from a single-stranded RNA template. It is a crucial tool in molecular biology and genetic research.
Automatically generated - may contain errors
Lab products found in correlation
Deoxynucleotide triphosphate, by Thermo Fisher Scientific (3 mentions)
Oligo dt primer, by Roche (3 mentions)
Platinum sybr green qpcr supermix udg kit, by Thermo Fisher Scientific (3 mentions)
Lightcycler system, by Roche (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
4 protocols using moloney murine leukemia virus m mulv reverse transcriptase
1
Osteoblast Gene Expression Analysis
2
Osteoclast Differentiation and Gene Expression Analysis
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Osteoclasts cells were seeded in 6-well plates for 7 days at 37 °C in 5 % CO2 in an osteoclast differentiation medium containing 50 ng/ml M-CSF and 50 ng/ml RANKL at a density of 1 × 106 cells/well and cells were treated with the TPF at concentrations of 0, 50, and 100 μg/ml. Total RNA from the cells of each well was isolated using NucleoSpin (Macherey–Nagel, Duren, Germany). RNA aliquots were reverse transcribed to complementary DNAs by using an oligo (dT) primer (Roche), deoxynucleotide triphosphate (dNTP), and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas, Hanover, MD, USA). The complementary DNA products were subjected to PCR amplification with gene-specific primers for mouse Cathepsin K, Mmp-9, and Mmp-13 (Table 1 ). Real-time RT-PCR amplification was performed using a Light Cycler System (Roche) with a Platinum SYBR Green qPCR Super Mix UDG kit (Invitrogen, Carlsbad, CA, USA).
Primer sequences of real-time PCR
| Gene | Forward | Reverse |
|---|---|---|
| TRAP | 5′CGTCTCTGCACAGATTGCAT | 3′AAGCGCAAACGGTAGTAAGG |
| Cathepsin K | 5′CGAAAAGAGCCTAGCGA | 3′TGGGTAGCAGCAGAAACA |
| MM9 | 5′GAACCAATCTCACCGACAGG | 3′GCCACCCGAGTGTAACCATA |
| MM13 | 5′GTCTGAGATTTGTAGGCCG | 3′TCATCAAGCTTCTGTCTGTGC |
3
RNA Isolation and qRT-PCR Analysis
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For ribonucleic acid (RNA) isolation, cell lysis was performed in the tissue culture plates using TRIzol reagent (Invitrogen), and subsequently subjected to deoxyribonuclease I (DNase-I) treatment. Using the respective oligo-deoxythymine (dT) primers (Invitrogen) and Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase (Fermentas), synthesis of the first-strand complementary DNA (cDNA) was conducted from the total RNA. Real-time PCR was performed using a QuantiTect SYBR Green PCR Kit (Qiagen). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. All samples were analyzed in triplicate according to the manufacturer’s instructions. The respective primer sequences are provided in the supplementary data (Table S1 ).
4
Osteoblast Gene Expression Analysis
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Osteoblasts cells were seeded in 6-well plates at a density of 1 × 106 cells/well. After 6-day culture, cells were treated with TPFs at concentrations of 0, 50, and 100 ng/ml for 48 h. Total RNA from the cells of each well was isolated, respectively using NucleoSpin (Macherey–Nagel, Duren, Germany). RNA aliquots were reverse transcribed to complementary DNAs by using an oligo (dT) primer (Roche), deoxynucleotide triphosphate (dNTP), and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas, Hanover, MD, USA). The complementary DNA products were subjected to PCR amplification with gene-specific primers for mouse osteocalcin, Runx2, osterix, Alp, Bmp-2, Bmp-4, and Bmp-7 (Table 1 ). Real-time RT-PCR amplification was performed using a LightCycler System (Roche) with a Platinum SYBR Green qPCR SuperMix UDG kit (Invitrogen, Carlsbad, CA, USA).
Primer sequences of real-time PCR
| Gene | Forward | Reverse |
|---|---|---|
| Alkaline phosphatase | 5′ ACAGCCATCCTGTATGGCAA | 3′ GCCTGGTAGTTGTTGTGAGCA |
| Osteocalcin | 5′ TGAGGACCATCTTCTGCTCA | 3′ TGGACATGAAGGCTTTGTCA |
| Osterix | 5′ TATGCTCCGACCTCCTCAACT | 3′ TCCTATTTGCCGTTTTCCCGA |
| Runx2 | 5′ GATCTGAGATTTGTAGGCCG | 3′ TCATCAAGCTTCTGTCTGTGCC |
| BMP-2 | 5′ CGGACTGCGGTCTCCTAA | 3′ GGGAAGCAGCAACACTAGA |
| BMP-4 | 5′ GACTTCGAGGCGACACTTCT | 3′ GCCGGTAAAGATCCCTCATGTA |
| BMP-7 | 5′ GAAAACAGCAGCAGTGACCA | 3′ GGTGGCGTTCATGTAGGAGT |
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