Total RNA was extracted from tissues using TRIzol (Invitrogen), and cDNA was then synthesized. Real-time PCR was performed according to the manufacturer’s instructions using a SYBR Premix Ex Taq (Takara Bio Inc.). For analysis, target gene expression was normalized to the housekeeping gene β-actin. Gene expression values were then calculated using the mean from the control samples as a calibrator. Real-time PCR primers were synthesized by Sangon Biotech (Table S1).
Real time pcr primers
Manufactured by Sangon
Sourced in China
Real-time PCR primers are short DNA sequences used in the amplification and detection of specific genetic targets during real-time polymerase chain reaction (qPCR) experiments. These primers are designed to bind to complementary regions of the target DNA, initiating the replication process that is monitored in real-time to quantify the amount of the target present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Cck 8, by Beyotime (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Ang 2, by Yuanye Bio-Technology (1 mentions)
Rt qpcr kit, by Takara Bio (1 mentions)
Sybr premix ex tap kit, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Random primer, by Sangon (1 mentions)
M mlv reverse transcriptase, by BioTeke (1 mentions)
Tripure kit, by BioTeke (1 mentions)
Oligo dt, by Sangon (1 mentions)
Power taq pcr mastermix, by BioTeke (1 mentions)
Sybr green, by Solarbio (1 mentions)
Alkaline phosphatase assay kit colorimetric, by Abcam (1 mentions)
α sma, by Cell Signaling Technology (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Glutamine, by Merck Group (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Tripolyphosphate tpp, by Merck Group (1 mentions)
Hsa mir 21 mimics, by GenePharma (1 mentions)
5 protocols using real time pcr primers
1
Real-Time PCR Gene Expression Analysis
2
Angiotensin II Signaling Pathway Protocol
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LIQ (CAS No. 551-15-5; purity ≥ 98%), Ang II was sourced from Shanghai YuanYe Biotechnology (Shanghai, China). Primary antibodies including anti-TAK1, anti-phos-JNK1/2, and anti-JNK1/2 were purchased from CST (Boston, USA). Primary antibodies including anti-ATE1 and secondary antibodies were purchased from Abcam (Cambridge, England); anti-phos-TAK1 was purchased from Sabbiotech (Maryland, USA). CCK-8 was purchased from Beyotime Biotechnology (Shanghai, China). The Phalloidin Staining Kit was purchased from Solarbio (Beijing, China). The RT-qPCR kit, Trizol, and the SYBR Premix Ex Tap kit were purchased from Takara (Japan). Real-time PCR primers were obtained from Sangon Biotech (Shanghai, China). All other chemicals used were sourced from Thermo Fisher Scientific (Pittsburgh, PA, USA).
3
RNA Extraction and qPCR for PPARγ Expression
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The total RNA was extracted with a TRIpure kit (BioTeke, Beijing, China) and reversely transcribed into cDNA with M-MLV reverse transcriptase (BioTeke) in the presence of Oligo(dT) and random primers (Sangon, Shanghai, China).
After concentration measurement, the cDNA was used for real-time PCR with 2×Power Taq PCR Master Mix (BioTeke) and SYBR Green (Solarbio, Beijing, China) to detect the mRNA level of PPARγ, with β-actin as the internal control. The procedure was set as follow: 94°C for 5 min, 94°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec, followed with 40 cycles of 72°C for 2 min 30 sec, 40°C for 1 min 30 sec, melting from 60°C to 94°C each 1°C for 1 sec, and finally incubated at 25°C for several minutes. The real-time PCR primers were purchased from Sangon, and the sequence information are shown inTable 1 . The data was analyzed with 2−ΔΔCt method.
After concentration measurement, the cDNA was used for real-time PCR with 2×Power Taq PCR Master Mix (BioTeke) and SYBR Green (Solarbio, Beijing, China) to detect the mRNA level of PPARγ, with β-actin as the internal control. The procedure was set as follow: 94°C for 5 min, 94°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec, followed with 40 cycles of 72°C for 2 min 30 sec, 40°C for 1 min 30 sec, melting from 60°C to 94°C each 1°C for 1 sec, and finally incubated at 25°C for several minutes. The real-time PCR primers were purchased from Sangon, and the sequence information are shown in
4
Osteogenic Differentiation Protocol
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Cell culture media were purchased from Invitrogen Technologies (Carlsbad, CA, USA). Real‐time PCR primers were purchased from Sangon Biotech (Shanghai, China). The miScript SYBR Green PCR kit was purchased from QIAGEN (Hilden, Germany). The Alkaline Phosphatase Colorimetric Assay Kit was purchased from Biovision (Milpitas, CA, USA). The Calcio Arsenazo III Proced was purchased from STANBIO laboratory (Boerne, TX, USA). The Pierce ECL Western Blotting KIT was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Specific antibodies against Runx2, SM22α, α‐SMA, and OC were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phosphorylated ERK1/2 and total ERK were obtained from Santa Cruz Technology (Santa Cruz, CA, USA). Specific β‐actin antibody was purchased from Sigma‐Aldrich. All secondary antibodies were purchased from Thermo Scientific (Waltham, MA, USA).
5
Chitosan-Based Composite Biomaterial
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Chitosan (CS) of 100 kDa with a deacetylation degree of 90%was purchased from Jinke Co., Ltd. (Zhejiang, China). Tripolyphosphate (TPP) and gelatine powder (type A) were obtained from Sigma (St. Louis, USA). Hyaluronic acid (HA) of 10 kDa was purchased from C.P. Freda Pharmaceuticals Ltd. (Shangdong, China) The hsa-miR-21 mimics (5’-uagcuuaucagacugauguugadTdT-3’; 5’-ucaacaucagucugauaagcuadTdT-3’) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Titanium substrates 1.0 mm thick and 14 mm in diameter (99.9%) were fabricated by the Northwest Institute for Nonferrous Metal Research (Xi’an, China). Collagenase type I, dispase and glutamine were purchased from Sigma (St. Louis, USA). Foetal bovine serum and α-MEM were provided by HyClone (MA, USA). The LDH cytotoxicity assay kit and CCK-8 were produced by Beyotime (Jiangsu, China). Real-time PCR primers were provided by Shanghai Sangon (Shanghai, China).
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