Abstract
BackgroundQuantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental conditions. No studies have validated specific reference genes in canine osteosarcoma (OS). Previous gene expression studies involving canine OS have used one or two reference genes to normalize gene expression. This study aimed to validate a panel of reference genes commonly used for normalization of canine OS gene expression data using the geNorm algorithm. qPCR analysis of nine canine reference genes was performed on 40 snap-frozen primary OS tumors and seven cell lines.ResultsTumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively.ConclusionsAppropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.
Highlights
Quantitative PCR is a common method for quantifying messenger RNA (mRNA) expression
Reference genes stably expressed in canine soft tissue sarcoma are β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6); while in canine mammary gland tumors were a combination of hypoxanthine-phosphoribosyl transferase, ATP-synthase subunit 5B, ribosomal protein L32 and ubiquitin
This study investigated the reliability of several reference genes expression in snap-frozen tumors and in cell lines of canine OS origin
Summary
Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental conditions. QPCR analysis of nine canine reference genes was performed on 40 snap-frozen primary OS tumors and seven cell lines. Gene expression studies in canine OS are valuable, as dogs qPCR is a sensitive method for quantifying mRNA gene transcripts; the two most popular real-time assays use SYBR® green fluorescent dye and the Taqman® probe. Many reports have demonstrated the importance of studying gene expression at the mRNA transcription level using snap-frozen tissues, micro-dissected tumors. The quantification of gene expression using the qPCR method requires appropriate standardization from initial tissue sampling, RNA extraction protocols, cDNA synthesis, assay characteristics, and reference gene validation [6, 7]. A reference gene should be stably expressed in tissues or cells regardless of the histology, pathological condition, or cellular physiological-metabolic state
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