Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

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Abstract

Background

Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR.

Results

In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin ( ACTB), beta-2-microglobulin ( B2M), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), hydroxymethylbilane synthase ( HMBS), hypoxanthine phosphoribosyltransferase 1 ( HPRT1), ribosomal protein L4 ( RPL4), succinate dehydrogenase complex subunit A ( SDHA), TATA box binding protein ( TPB)and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide ( YWHAZ). The stability of these reference genes in different pig tissues was investigated using the geNorm application. The range of expression stability in the genes analysed was (from the most stable to the least stable): ACTB/ RPL4, TBP, HPRT, HMBS, YWHAZ, SDHA, B2M and GAPDH.

Conclusion

Expression stability varies greatly between genes. ACTB, RPL4, TPB and HPRT1 were found to have the highest stability across tissues. Based on both expression stability and expression level, our data suggest that ACTB and RPL4 are good reference genes for high abundant transcripts while TPB and HPRT1 are good reference genes for low abundant transcripts in expression studies across different pig tissues.

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Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

Nicholas Silver, Steve Best, Jie Jiang … (2006)

Background Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Results Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Conclusion Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.

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Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes.

E T Waas, Dorine W. Swinkels, Paul N. Span … (2004)

For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, beta-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, beta-2-microglobin, beta-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.

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beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.

Julia Bailey-Serres, E Glare, Maaike E. Walters … (2002)

The use of reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels has led to the common use of beta-actin and GAPDH housekeeping genes as denominators for comparison of samples. Expression of these genes is assumed to remain constant, so normalising for variations in processing and signal quantitation. However, it is well documented that beta-actin and GAPDH expression is upregulated with proliferation, activation, and differentiation. We hypothesised that airway samples which differ in their cellular profiles and activation status have different levels of expression of GAPDH and beta-actin. The mRNA for beta-actin, GAPDH, and interleukin (IL)-2 was measured in bronchoalveolar lavage (BAL) fluid cells and endobronchial biopsy tissue by competitive RT-PCR in a cross sectional study of 26 normal controls and 92 asthmatic subjects. For both BAL fluid cells and biopsy tissue, asthmatics overall had reduced expression of GAPDH and beta-actin mRNA. In asthmatic subjects not using inhaled corticosteroids (ICS), GAPDH mRNA levels in both BAL fluid and biopsy tissue, and beta-actin mRNA in BAL fluid cells were 10 times lower than samples from both normal controls and from asthmatic subjects using ICS. beta-Actin mRNA in biopsy specimens showed the same pattern of expression, but asthmatic subjects not using ICS were not significantly different from those receiving ICS treatment. IL-2 mRNA levels did not differ between the subject or treatment groups but, when expressed as a ratio with beta-actin, significant differences were seen. beta-Actin and GAPDH used as denominators of gene expression quantitation in asthma research can cause confounding. Housekeeping genes need careful validation before their use in such quantitative mRNA assays.

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Author and article information

Journal

Journal ID (nlm-ta): BMC Mol Biol

Title: BMC Molecular Biology

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2199

Publication date Collection: 2007

Publication date (Electronic): 15 August 2007

Volume: 8

Page: 67

Affiliations

[1 ]University of Copenhagen, Faculty of Life Sciences, Department of Basic Animal and Veterinary Sciences, Division of Genetics and Bioinformatics, Groennegaardsvej 3, 1870 Frederiksberg C, Denmark

Article

Publisher ID: 1471-2199-8-67

DOI: 10.1186/1471-2199-8-67

PMC ID: 2000887

PubMed ID: 17697375

SO-VID: 291346c8-5096-4206-91c2-5d3848e0571f

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

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Most cited references 17

Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes.

beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.

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