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Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint
Published on: July 22, 2021
Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from
Lindsey J Maccoux1, Dylan N Clements, Fiona Salway
1Centre for Integrated Genomic Medical Research, University of Manchester, The Stopford Building, Manchester, UK. lmaccoux@googlemail.com <lmaccoux@googlemail.com>
BMC Molecular Biology
|July 27, 2007
Summary
Identifying stable reference genes for gene expression analysis is crucial. This study found mitochondrial ribosomal protein S7 (MRPS7) to be a highly stable reference gene across canine articular tissues, outperforming GAPDH.
Area of Science:
- Molecular Biology
- Genomics
Background:
- Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is essential for accurate gene expression measurement.
- Gene expression data normalization requires stable reference genes, but universally optimal ones are lacking.
- Existing reference genes may not be suitable across diverse tissue types or disease states.
Purpose of the Study:
- To identify novel, stably expressed reference genes for gene expression analysis in canine articular tissues.
- To evaluate the expression stability of candidate reference genes across different articular tissue types (bone, ligament, cartilage, synovium, fat).
- To compare the performance of newly identified reference genes against commonly used ones like GAPDH.
Main Methods:
- Microarray data analysis was used to identify potential reference genes from normal and osteoarthritic canine articular tissues.
- Real-time RT-qPCR assays were developed and performed for selected candidate genes.
- Reference gene expression stability was assessed using three distinct mathematical algorithms (Bestkeeper, geNorm, and conventional normalization).
Main Results:
- Twelve novel reference genes were identified from microarray data.
- Mitochondrial ribosomal protein S7 (MRPS7) exhibited stable expression across all five evaluated canine articular tissues.
- The commonly used reference gene GAPDH showed unstable expression in all evaluated tissues.
- Newly identified reference genes, validated via conventional normalization, demonstrated greater stability than those identified using real-time RT-qPCR normalization.
Conclusions:
- A stepwise filtering procedure applied to conventionally normalized microarray data effectively identifies novel, stable reference genes.
- Mitochondrial ribosomal protein S7 (MRPS7) is a promising reference gene for future studies in canine tissues and diseases.
- Different stability assessment algorithms generally agree on the most and least stable reference genes, particularly when gene co-regulation is absent.