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How are genes measured? Examples from studies on iron metabolism in pregnancy

Published online by Cambridge University Press:  07 March 2007

Henriette S. Andersen  and
Harry J. McArdle
Henriette S. Andersen*
Affiliation:
Development, Growth and Function Division, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
Harry J. McArdle
Affiliation:
Development, Growth and Function Division, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK
*
Corresponding author: Dr Henriette S. Andersen, fax +44 1224 716622, email [email protected]
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Abstract

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As the 21st century moves forward, it is becoming more and more apparent that the genetic makeup of any individual strongly influences the way they metabolise nutrients. It is very important, therefore, to understand the techniques and technologies used to assess the contribution genes make to the physiology of an individual. Clearly, it is not possible to provide a comprehensive overview, but in the present review an attempt will be made to show, using examples from the authors' research, how these methods have contributed to this understanding. Studies are being undertaken into Fe transport across the placenta, from the mother to the fetus, and the consequences of maternal anaemia on pregnancy outcome. Levels of gene transcript and protein have been measured using Northern and Western blotting respectively. During the course of this work a new protein has been identified using the available human genome database. Following this ‘in silico’ or ‘cyber biology’, techniques such as real-time RT–PCR and RNA interference have been used to examine expression of this gene and its protein. The methods used, briefly how they work and some of their limitations will be explained. The objective of the present review is primarily to give a better perception of how molecular biology can be used in research and to help gain a clearer understanding of some of the techniques used.

Keywords

Real-time RT–PCRShort interfering RNAMulti-Cu ferroxidase
Type
Symposium on ‘How and why measure individual variability’
Information
Proceedings of the Nutrition Society , Volume 63 , Issue 3 , August 2004 , pp. 481 - 490
Copyright
Copyright © The Nutrition Society 2004

References

Benson, DA, Boguski, MS, Lipman, DJ, Ostell, J, Ouellette, BF, Rapp, BA & Wheeler, DL (1999) GenBank. Nucleic Acids Research 27, 38–43.Google Scholar
Bradford, MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.Google Scholar
Brummelkamp, TR, Bernards, R & Agami, R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.Google Scholar
Bustin, SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25, 169–193.CrossRefGoogle ScholarPubMed
Caplen, NJ, Parrish, S, Imani, F, Fire, A & Morgan, RA (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proceedings of the National Academy of Sciences USA 98, 9742–9747.Google Scholar
Carlberg, C (1999) Lipid soluble vitamins in gene regulation. Biofactors 10, 91–97.Google Scholar
Clarke, SD & Abraham, S (1992) Gene expression: nutrient control of pre- and posttranscriptional events. FASEB Journal 6, 3146–3152.CrossRefGoogle ScholarPubMed
Clarke, SD, Gasperikova, D, Nelson, C, Lapillonne, A & Heird, WC (2002) Fatty acid regulation of gene expression: a genomic explanation for the benefits of the mediterranean diet. Annals of New York Academy of Sciences 967, 283–298.Google Scholar
Claverie, JM (1999) Computational methods for the identification of differential and coordinated gene expression. Human Molecular Genetics 8, 1821–1832.CrossRefGoogle ScholarPubMed
Conklin, BS, Zhong, DS, Zhao, W, Lin, PH & Chen, C (2002) Shear stress regulates occludin and VEGF expression in porcine arterial endothelial cells. Journal of Surgical Research 102, 13–21.Google Scholar
Danzeisen, R, Fosset, C, Page, K, David, S & McArdle, HJ (2002) Placental ceruloplasmin homologue is regulated by iron and copper and is implicated in iron metabolism. American Journal of Physiology 282, C472–C478.Google Scholar
Danzeisen, R, Ponnambalam, SV & McArdle, HJ (2000) The effect of ceruloplasmin on iron release from placental (BeWo) cells: evidence for an endogenous Cu oxidase. Placenta 21, 805–812.CrossRefGoogle ScholarPubMed
Donovan, A, Brownlie, A, Zhou, Y, Shepard, J, Pratt, SJ, Moynihan, J, Paw, BH, Drejer, A, Barut, B, Zapata, A, Law, TC, Brugnara, C, Lux, SE, Pinkus, GS, Pinkus, JL, Kingsley, PD, Palis, J, Fleming, MD, Andrews, NC & Zon, LI (2000) Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 403, 776–781.Google Scholar
Donze, O & Picard, D (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Research 30, 2–4.Google Scholar
Dykxhoorn, DM, Novina, CD & Sharp, PA (2003) Killing the messenger: short RNAs that silence gene expression. Nature Reviews Molecular Cell Biology 4, 457–467.CrossRefGoogle ScholarPubMed
Elbashir, SM, Harborth, J, Lendeckel, W, Yalcin, A, Weber, K & Tuschl, T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.Google Scholar
Elbashir, SM, Harboth, J, Weber, K & Tuschl, T (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213.CrossRefGoogle ScholarPubMed
Emmert-Buck, MR, Bonner, RF, Smith, PD, Chuaqui, RF, Zhuang, Z, Goldstein, SR, Weiss, RA & Liotta, LA (1996) Laser capture microdissection. Science 274, 998–1001.Google Scholar
Fire, A, Xu, S, Montgomery, MK, Kostas, SA, Driver, SE & Mello, CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.CrossRefGoogle ScholarPubMed
Fryer, AA, Jones, P, Strange, R, Hume, R & Bell, JE (1993) Plasma protein levels in normal human fetuses: 13 to 41 weeks' gestation. British Journal of Obstetrics and Gynecology 100, 850–855.CrossRefGoogle ScholarPubMed
Gambling, L, Danzeisen, R, Gair, S, Lea, RG, Charania, Z, Solanky, N, Joory, KD, Srai, SK & McArdle, HJ (2001) Effect of iron deficiency on placental transfer of iron and expression of iron transport proteins in vivo and in vitro. Biochemical Journal 356, 883–889.CrossRefGoogle ScholarPubMed
Gersten, DM (1996) Gel Electrophoresis. West Sussex: John Wiley & Sons Ltd.Google Scholar
Ginzinger, DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Experimental Hematology 30, 503–512.Google Scholar
Hammond, SM, Caudy, AA & Hannon, GJ (2001) Post-transcriptional gene silencing by double-stranded RNA. Nature Reviews Genetics 2, 110–119.CrossRefGoogle ScholarPubMed
Hannon, GJ (2002) RNA interference. Nature 418, 244–251.Google Scholar
Harborth, J, Elbashir, SM, Bechert, K, Tuschl, T & Weber, K (2001) Identification of essential genes in cultured mammalian cells using small interfering RNAs. Journal of Cell Science 114, 4557–4565.Google Scholar
Harris, ZL, Durley, AP, Man, TK & Gitlin, JD (1999) Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proceedings of the National Academy of Sciences USA 96, 10812–10817.CrossRefGoogle ScholarPubMed
Higuchi, R, Fockler, C, Dollinger, G & Watson, R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026–1030.Google Scholar
Hu, A, Colella, M, Tam, JS, Rappaport, R & Cheng, SM (2003) Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR. Journal of Clinical Microbiology 41, 149–154.CrossRefGoogle Scholar
Jennings, EG & Young, RA (1999) Genome expression on the World Wide Web. Trends in Genetics 15, 202–204.CrossRefGoogle ScholarPubMed
Jones, LJ, Haugland, RP & Singer, VL (2003) Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution. Biotechniques 34, 850–854, 856, 858.CrossRefGoogle ScholarPubMed
Kisielow, M, Kleiner, S, Nagasawa, M, Faisal, A & Nagamine, Y (2002) Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA. Biochemical Journal 363, 1–5.CrossRefGoogle ScholarPubMed
Lekanne Deprez, RH, Fijnvandraat, AC, Ruijter, JM & Moorman, AF (2002) Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Analytical Biochemistry 307, 63–69.CrossRefGoogle ScholarPubMed
McArdle, HJ, Danzeisen, R, Fosset, C & Gambling, L (2003) The role of the placenta in iron transfer from mother to fetus and the relationship between iron status and fetal outcome. Biometals 16, 161–167.Google Scholar
McArdle, HJ, Douglas, AJ, Bowen, BJ & Morgan, EH (1985) The mechanism of iron uptake by the rat placenta. Journal of Cell Physiology 124, 446–450.Google Scholar
McArdle, HJ, Douglas, AJ & Morgan, EH (1984) Transferrin binding by microvillar vesicles isolated from rat placenta. Placenta 5, 131–138.Google Scholar
McArdle, HJ & Morgan, EH (1982) Transferrin and iron movements in the rat conceptus during gestation. Journal of Reproduction and Fertility 66, 529–536.Google Scholar
McArdle, HJ & Morgan, EH (1984) The effect of monoclonal antibodies to the human transferrin receptor on transferrin and iron uptake by rat and rabbit reticulocytes. Journal of Biological Chemistry 259, 1398–1400.CrossRefGoogle Scholar
McArdle, HJ & Tysoe, J (1988) Effect of nicotine on transferrin binding and iron uptake by cultured rat placenta. Journal of Cell Physiology 134, 509–513.Google Scholar
Mullis, KB (1990) Target amplification for DNA analysis by the polymerase chain reaction. Annales de Biologie Clinique 48, 579–582.Google Scholar
Nadeau, JH & Dunn, PJ (1998) Genomic strategies for defining and dissecting development and physiological pathways. Current Opinion in Genetic Development 8, 311–315.Google Scholar
Quail, EA & Yeoh, GC (1995) The effect of iron status on glyceraldehyde 3-phosphate dehydrogenase expression in rat liver. FEBS Letters 13, 126–128.Google Scholar
Roeser, HP, Lee, GR, Nacht, S & Cartwright, GE (1970) The role of ceruloplasmin in iron metabolism. Journal of Clinical Investigation 49, 2408–2417.CrossRefGoogle ScholarPubMed
Rottman, JB (2002) The ribonuclease protection assay: a powerful tool for the veterinary pathologist. Veterinary Pathology 39, 2–9.CrossRefGoogle ScholarPubMed
Saiki, RK, Bugawan, TL, Horn, GT, Mullis, KB & Erlich, HA (1986) Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 324, 163–166.Google Scholar
Saiki, RK, Gelfand, DH, Stoffel, S, Scharf, SJ, Higuchi, R, Horn, GT, Mullis, KB & Erlich, HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.Google Scholar
Schmittgen, TD & Zakrajsek, BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. Journal of Biochemical and Biophysical Methods 46, 69–81.Google Scholar
Selvey, S, Thompson, EW, Matthaei, K, Lea, RA, Irving, MG & Griffiths, LR (2001) Beta-actin – an unsuitable internal control for RT-PCR. Molecular Cell Probes 15, 307–311.Google Scholar
Sorensen, DR, Leirdal, M & Sioud, M (2003) Gene silencing by systemic delivery of synthetic siRNAs in adult mice. Journal of Molecular Biology 327, 761–766.Google Scholar
Templeton, DM & Liu, Y (2003) Genetic regulation of cell function in response to iron overload or chelation. Biochimica et Biophysica Acta 1619, 113–124.Google Scholar
Thomas, PS (1980) Hybridisation of denatured RNA and small DNA fragments transferred to nitrocellulose. Proceedings of the National Academy of Sciences USA 77, 5201–5205.Google Scholar
Towbin, H, Staehelin, T & Gordon, J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA 76, 4350–4354.Google Scholar
Tricarico, C, Pinzani, P, Bianchi, S, Paglierani, M, Distante, V, Pazzagli, M, Bustin, SA & Orlando, C (2002) Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Analytical Biochemistry 309, 293–300.Google Scholar
Turchin, A & Kohane, IS (2002) Gene homology resources on the World Wide Web. Physiological Genomics 11, 165–177.Google Scholar
Vaucheret, H, Beclin, C & Fagard, M (2001) Post-transcriptional gene silencing in plants. Journal of Cell Science 114, 3083–3091.Google Scholar
Vulpe, CD, Kuo, YM, Murphy, TL, Cowley, L, Askwith, C, Libina, N, Gitschier, J & Anderson, GJ (1999) Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nature Genetics 21, 195–199.Google Scholar
Walmsley, ME & Patient, RK (1994) Quantitative and qualitative analysis of exogenous gene expression by the S1 nuclease protection assay. Molecular Biotechnology 3, 265–275.Google Scholar
Wittwer, CT, Herrmann, MG, Gundry, CN & Elenitoba-Johnson, KSJ (2001) Real-time multiplex PCR assays. Methods 25, 430–442.Google Scholar