Primary chondrocytes were prepared from the dissected articular cartilage and cultured as discussed above. Total RNA was isolated from the chondrocytes using Trizol (Invitrogen, Burlington, Ontario, Canada). RNeasy Mini kit (QIAGEN, Toronto, Ontario, Canada) was used, including on-column DNase digestion to eliminate DNA (RNase-Free DNase Set, QIAGEN). RNA were reverse transcribed and amplified using the QuantiTect Reverse Transcription PCR Kit (QIAGEN) on the Rotor Gene 3000 real-time PCR system (Corbett Research, Mortlake, Australia) according to the manufacturer’s protocol as shown before.6 Fold increase in PCR products was analysed using a 2−ΔΔCt method. All experiments were performed in duplicate for each sample, and the primers were designed using Primer3 online software. Data were normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) levels and represent averages and SEM. The statistical significance of quantitative polymerase chain reaction (qPCR) results was determined by a two-way analysis of variance with the Bonferroni post-test, using GraphPad Prism V.3.00 for Windows.
Quantitect reverse transcription pcr kit
Manufactured by Qiagen
Sourced in Australia
The QuantiTect Reverse Transcription PCR Kit is a laboratory equipment product designed for reverse transcription and real-time PCR of RNA samples. It enables efficient conversion of RNA to cDNA and subsequent amplification and quantification of target sequences.
Automatically generated - may contain errors
2 protocols using quantitect reverse transcription pcr kit
1
Chondrocyte RNA Extraction and qPCR Analysis
2
Analyzing Knee Cartilage Gene Expression
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Knee cartilage specimens from WT and 12/15-LOXÀ/À mice at the ages of 3 and 6 months (n ¼ 5 per time point per genotype), and from sham and DMM-operated mice (n ¼ 5 per time point per genotype) at 2 weeks post-surgery were collected, and pooled together for the isolation of total RNA using the TRIzol ® reagent (Invitrogen, Burlington, ON, Canada), according to the manufacturer's instructions. RNA was reverse transcribed and amplified using the QuantiTect Reverse Transcription PCR Kit (QIAGEN) on the Rotor Gene 3000 realtime PCR system (Corbett Research, Mortlake, Australia) according to the manufacturer's protocol. Relative messenger RNA (mRNA) expression was determined using the DDC T method and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. Each PCR was performed in triplicate from two independent experiments.
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