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Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves.

ABSTRACT: Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.

SUBMITTER: Gambarotta G

PROVIDER: S-EPMC4140797 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves.

Gambarotta Giovanna G Ronchi Giulia G Friard Olivier O Galletta Pantaleo P Perroteau Isabelle I Geuna Stefano S

PloS one 20140821 8

Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new ...[more]

PMID: 25144298

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Project description:PurposeTo evaluate the suitability of common housekeeping genes (HKGs) for use in quantitative reverse transcription PCR (qRT-PCR) assays of the cornea in various murine disease models.MethodsCORNEAL DISEASE MODELS STUDIED WERE: 1) corneal neovascularization (CorNV) induced by suture or chemical burn, 2) corneal infection with Candida albicans or Aspergillus fumigatus by intrastromal injection of live spores, and 3) perforating corneal injury (PCI) in Balb/c mice or C57BL/6 mice. Expression of 8 HKGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], beta-actin [ACTB], lactate dehydrogenase A [LDHA], ribosomal protein L5 [RPL5], ubiquitin C [UBC], peptidylprolyl isomerase A [PPIA], TATA-box binding protein [TBP1], and hypoxanthine guanine phosphoribosyl transferase [HPRT1]) in the cornea were measured at various time points by microarray hybridization or qRT-PCR and the data analyzed using geNorm and NormFinder.ResultsMicroarray results showed that under the CorNV condition the expression stability of the 8 HKGs decreased in order of PPIA>RPL5>HPRT1>ACTB>UBC>TBP1>GAPDH>LDHA. qRT-PCR analyses demonstrated that expression of none of the 8 HKGs remained stable under all conditions, while GAPDH and ACTB were among the least stably expressed markers under most conditions. Both geNorm and NormFinder analyses proposed best HKGs or HKG combinations that differ between the various models. NormFinder proposed PPIA as best HKG for three CorNV models and PCI model, as well as UBC for two fungal keratitis models. geNorm analysis demonstrated that a similar model in different mice strains or caused by different stimuli may require different HKGs or HKG pairs for the best normalization. Namely, geNorm proposed PPIA and HRPT1 and PPIA and RPL5 pairs for chemical burn-induced CorNV in Balb/c and C57BL/6 mice, respectively, while UBC and HPRT1 and UBC and LDHA were best for Candida and Aspergillus induced keratitis in Balb/c mice, respectively.ConclusionsWhen qRT-PCR is designed for studies of gene expression in murine cornea, preselection of situation-specific reference genes is recommended. In the absence of knowledge about situation-specific HKGs, PPIA and UBC, either alone or in combination with HPRT1 or RPL5, can be employed.

Project description:BACKGROUND: Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats. RESULTS: RNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), beta-actin (ACTB), beta-2-microglobulin (B2M), beta-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones. CONCLUSION: The reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.

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Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves.

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Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves.

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