Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR.

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
Read Online Read More Add to Saved list
  • Author(s): Rho HW;Rho HW; Lee BC; Choi ES; Choi IJ; Lee YS; Goh SH
  • Source:
    BMC cancer [BMC Cancer] 2010 May 28; Vol. 10, pp. 240. Date of Electronic Publication: 2010 May 28.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't; Validation Study
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: BioMed Central Country of Publication: England NLM ID: 100967800 Publication Model: Electronic Cited Medium: Internet ISSN: 1471-2407 (Electronic) Linking ISSN: 14712407 NLM ISO Abbreviation: BMC Cancer Subsets: MEDLINE
    • Publication Information:
      Original Publication: London : BioMed Central, [2001-
    • Subject Terms:
      Gene Expression Profiling*/standards ; Genetic Markers* ; Reverse Transcriptase Polymerase Chain Reaction*/standards; Blood Coagulation Factors/*genetics ; Glyceraldehyde-3-Phosphate Dehydrogenases/*genetics ; Stomach Neoplasms/*genetics ; beta 2-Microglobulin/*genetics; Algorithms ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; RNA Stability ; RNA-Binding Proteins ; Reference Standards ; Reproducibility of Results ; Ribosomal Proteins
    • Abstract:
      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.
    • References:
      BMC Cancer. 2009 Feb 06;9:49. (PMID: 19200351)
      BMC Mol Biol. 2008 Jul 08;9:62. (PMID: 18611280)
      World J Surg Oncol. 2003 Dec 19;1(1):28. (PMID: 14683528)
      PLoS Med. 2005 Feb;2(2):e45. (PMID: 15737001)
      Physiol Genomics. 2004 Jul 08;18(2):226-31. (PMID: 15161965)
      Mol Aspects Med. 2006 Apr-Jun;27(2-3):126-39. (PMID: 16469371)
      Anal Biochem. 2009 Nov 1;394(1):110-6. (PMID: 19622337)
      PLoS One. 2009 Jul 07;4(7):e6162. (PMID: 19584937)
      Cancer Lett. 2005 Sep 28;227(2):185-91. (PMID: 16112421)
      Cancer Res. 2004 Aug 1;64(15):5245-50. (PMID: 15289330)
      Clin Chem. 2009 Apr;55(4):611-22. (PMID: 19246619)
      BMC Cancer. 2008 Jan 22;8:20. (PMID: 18211679)
      Genome Biol. 2002 Jun 18;3(7):RESEARCH0034. (PMID: 12184808)
      J Urol. 2006 May;175(5):1915-20. (PMID: 16600798)
      Anal Biochem. 1999 May 15;270(1):41-9. (PMID: 10328763)
      BMC Cancer. 2006 Jul 26;6:200. (PMID: 16872493)
      BMC Mol Biol. 2005 Feb 18;6:4. (PMID: 15720708)
      Breast Cancer Res. 2007;9(5):R58. (PMID: 17845721)
      J Mol Med (Berl). 2005 Dec;83(12):1014-24. (PMID: 16211407)
      Thorax. 2002 Sep;57(9):765-70. (PMID: 12200519)
      Mol Hum Reprod. 2003 Jul;9(7):411-20. (PMID: 12802048)
      BMC Mol Biol. 2008 May 06;9:46. (PMID: 18460208)
      Cell Cycle. 2005 Feb;4(2):315-22. (PMID: 15684612)
      Biotechnol Lett. 2004 Mar;26(6):509-15. (PMID: 15127793)
      J Biochem Biophys Methods. 2000 Nov 20;46(1-2):69-81. (PMID: 11086195)
      CA Cancer J Clin. 2005 Mar-Apr;55(2):74-108. (PMID: 15761078)
      J Cancer Res Clin Oncol. 2008 Sep;134(9):979-86. (PMID: 18317805)
      BMC Mol Biol. 2007 Jun 08;8:47. (PMID: 17559644)
      BMC Cancer. 2008 Nov 27;8:350. (PMID: 19036168)
    • Accession Number:
      0 (Blood Coagulation Factors)
      0 (Genetic Markers)
      0 (RNA-Binding Proteins)
      0 (RPL29 protein, human)
      0 (Ribosomal Proteins)
      0 (beta 2-Microglobulin)
      EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
    • Publication Date:
      Date Created: 20100529 Date Completed: 20100816 Latest Revision: 20211020
    • Publication Date:
      20231215
    • Accession Number:
      PMC2887403
    • Accession Number:
      10.1186/1471-2407-10-240
    • Accession Number:
      20507635