The mRNA expression of OA and apoptosis-specific gene was detected by qRT-PCR. For the in vitro study, cells were washed once with PBS and the total RNA was extracted using an RNA isolation kit (Tiangen Biotechnology; Beijing, China) according to the manufacturer's instructions. For the in vivo investigation, cartilage obtained at 4 and 8 weeks post-treatment was pulverized in liquid nitrogen before RNA extraction. Approximately 300 ng of total RNA was used as a template and reverse transcribed into cDNA using a reverse transcription kit (Fermentas Company, USA). The qRT-PCR reactions were performed using a Quantitative PCR Detection System (Realplex 4, Eppendorf Corporation, USA) with FastStart Universal SYBR Green Master (Mix, Roche company, Germany) and at the conditions of 10 min at 95°C, 15 s at 95°C and 1 min at 60°C. The primers used for PCR are shown in Table 1. Each gene was analyzed in triplicate to diminish operation errors. The relative gene expression levels were calculated by using the 2 -ΔΔCt method and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal reference.
Quantitative pcr detection system realplex 4
Manufactured by Eppendorf
Sourced in United States
The Eppendorf Quantitative PCR Detection System (Realplex 4) is a real-time PCR instrument designed for quantitative gene expression analysis. It features four independent thermal cyclers and fluorescence detection channels for simultaneous analysis of multiple samples.
Automatically generated - may contain errors
Lab products found in correlation
Faststart universal sybr green master mix, by Roche (4 mentions)
Rna isolation kit, by Tiangen Biotech (3 mentions)
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Safranin o, by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Live dead viability assay kit, by Thermo Fisher Scientific (1 mentions)
Alpha modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Solarbio (1 mentions)
Upright microscope, by Olympus (1 mentions)
Penicillin, by Solarbio (1 mentions)
Streptomycin, by Solarbio (1 mentions)
Sybr green mix kit, by Roche (1 mentions)
4 protocols using quantitative pcr detection system realplex 4
1
Quantification of OA and Apoptosis Gene Expression
2
Quantitative Analysis of Extracellular Matrix
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The genetic information was detected by qRT-PCR for type I, II and X collagen, aggrecan, Sox9, MMP-1, MMP-13 and TIMP-1(the primer sequences were showed in Table 1
3
Chondrocyte Isolation and Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We purchased 0.25% trypsin and 1% (v/v) antibiotics (penicillin 100 U/mL and streptomycin 100 U/mL) from Solarbio company in China; COL1A1(collagen I) antibody, COL2A1 (collagen II) antibody, and 3, 3-diaminobenzidine tetrahydrochloride (DAB) kit from Boster company in China; alpha-modified Eagle's medium (α-MEM), 20% (v/v) foetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazolium-romide (MTT) from Gibco company in USA; safranin O, proteinase K, Hoechst 33258, and chondroitin sulphate from Sigma company in USA; and incubator and Multiskan GO Microplate Spectrophotometer from Thermo Fisher Scientific company in USA. Other reagents and instruments in this study are as follows: the RNeasy RNA extraction kit (Tiangen Biotechnology, Beijing, China); hematoxylin-eosin (HE) kit (Jiancheng Biotech, China); reverse transcription kit (Fermentas company, USA); SYBR-Green mix kit (Roche company, Germany); FastStart Universal SYBR Green Master Mix (Roche Company, Germany); Quantitative PCR Detection System (realplex 4, Eppendorf Corporation, USA); live-dead viability assay kit (Invitrogen, USA); laser scanning confocal microscope (Nikon A1, Japan); and upright microscope (Olympus, Japan).
4
Quantitative Expression Analysis of Cartilage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qRT-PCR was used to analyze the expression of type I, II and X collagen, aggrecan, Sox9, MMP-1, MMP-3 and TIMP-1. The sequences of the specific primers were designed according to specific genes and shown in Table 1 . Total intracellular RNA was extracted using RNA isolation kits (Tiangen Biotechnology; Beijing, China). Approximately 300 ng of total RNA was used as templates for the reverse transcription into cDNA using reverse transcription kit (Fermentas Company, Waltham, MA, USA). The qRT-PCR reactions were performed on a Quantitative PCR Detection System (Realplex 4, Eppendorf Corporation, Hauppauge, NY, USA) with a FastStart Universal SYBR Green Master (Mix, Roche Company, Grenzach, Germany) under the cycle conditions of 10 min at 95, 15 s at 95 °C and 1 min at 60 °C. The melting curve data were recorded to verify the PCR specificity. Each gene was analyzed in triplicate to diminish operation errors. The relative gene expression levels were calculated using the 2−ΔΔCt method according to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!