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Identification of valid endogenous control genes for determining gene expression in human glioma.

ABSTRACT: In human glioma, quantitative real-time reverse-transcription PCR (qPCR) is a frequently used research tool. However, no systematic analysis of suitable reference genes for reliable gene expression analysis has been performed so far. In the current study, we tested 19 commonly used reference genes for their expression stability in human astrocytoma WHO Grade II, astrocytoma WHO Grade III, and glioblastoma (WHO Grade IV) both alone and compared with normal brain. First, equivalence tests for equal expression of candidate genes were applied, and those genes showing differential expression were ruled out from further analyses. Second, expression stability of the remaining candidate genes was determined by the NormFinder software. Generally, glioblastoma exhibited the highest expression levels and largest variability of candidate genes, whereas the opposite was true for normal brain. Even though Normfinder analyses revealed a large number of genes suitable for normalization in each of the tumor subgroups and across these groups, this number was drastically reduced after inclusion of normal brain into the analyses: Only GAPDH, IPO8, RPL13A, SDHA, and TBP were expected not to be differentially expressed; NormFinder analysis indicated favorable stability values for all of these genes, with TBP and IPO8 being the most stable ones. These 5 genes represent different physiological pathways and may be regarded as universal reference genes applicable for accurate normalization of gene expression in human astrocytomas of different grades (WHO Grades II-IV) alone and compared with normal brain, thereby enabling longitudinally designed studies (eg, in astrocytoma before and after malignant transformation).

SUBMITTER: Kreth S

PROVIDER: S-EPMC2940642 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Identification of valid endogenous control genes for determining gene expression in human glioma.

Kreth Simone S Heyn Jens J Grau Stefan S Kretzschmar Hans A HA Egensperger Rupert R Kreth Friedrich W FW

Neuro-oncology 20100205 6

In human glioma, quantitative real-time reverse-transcription PCR (qPCR) is a frequently used research tool. However, no systematic analysis of suitable reference genes for reliable gene expression analysis has been performed so far. In the current study, we tested 19 commonly used reference genes for their expression stability in human astrocytoma WHO Grade II, astrocytoma WHO Grade III, and glioblastoma (WHO Grade IV) both alone and compared with normal brain. First, equivalence tests for equa ...[more]

PMID: 20511187

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Project description:BACKGROUND: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. METHODS: We assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. RESULTS: This RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination. CONCLUSION: This study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

Project description:PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. CONCLUSION: This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium.

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Identification of valid endogenous control genes for determining gene expression in human glioma.

Publications

Identification of valid endogenous control genes for determining gene expression in human glioma.

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