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Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.

ABSTRACT: BACKGROUND: Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses. RESULTS: In the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids. CONCLUSION: Based on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor.

SUBMITTER: Bogaert L

PROVIDER: S-EPMC1484482 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.

Bogaert Lies L Van Poucke Mario M De Baere Cindy C Peelman Luc L Gasthuys Frank F Martens Ann A

BMC biotechnology 20060427

<h4>Background</h4>Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it ...[more]

PMID: 16643647

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Project description:BackgroundRT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach.ResultsIn this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results.ConclusionOur study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.

Dataset Information

Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.

Publications

Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.

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