Gene Expression Analysis of RNA Purified from Embryonic Stem Cells and Embryoid Body–Derived Cells Using a High‐Throughput Microarray Platform

Audrey Player1, Yonghong Wang2, Mahendra Rao3, Ernest Kawasaki1

1 National Cancer Institute, Bethesda, Maryland, 2 SAIC‐Frederick, Inc., NCI‐Frederick, Frederick, Maryland, 3 Johns Hopkins University School of Medicine, Baltimore, Maryland and Invitrogen Corp. Carlsbad California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1B.2
DOI:  10.1002/9780470151808.sc01b02s2
Online Posting Date:  September, 2007
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Abstract

In this unit, starting with purified RNA, experimental protocols for performing microarrray expression analysis of embryonic stem cell lines compared to their corresponding differentiated embryoidal bodies are described. Methods for data analysis are suggested, with the goal of determining which genes are differentially expressed between the preparations. As an example, the use of the Affymetrix microarray expression platform is described, but alternative experimental options for analysis of RNA transcript levels are also summarized. This unit suggests quality control metrics, summazrizes the critical parameters necessary for obtaining reproducible experimental results, and outlines quantitative PCR methods for validating microarray results. Curr. Protoc. Stem Cell Biol. 2:1B.2.1‐1B.2.36. © 2007 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; embryoid bodies; RNA expression analysis; microarray platform

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generating cDNA and In Vitro–Transcription (IVT) aRNA
  • Support Protocol 1: Assessment of ESC and EB RNA Quality Using the Agilent Bioanalyzer
  • Alternate Protocol 1: Two‐Cycle aRNA Amplification
  • Basic Protocol 2: Hybridizing, Washing, Staining, and Scanning of the Genechip
  • Basic Protocol 3: Data Analysis Using GCOS Batch Analysis Tool
  • Alternate Protocol 2: Data Analysis Using Modified PM‐Only Method
  • Basic Protocol 4: Real‐Time PCR Validation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generating cDNA and In Vitro–Transcription (IVT) aRNA

  Materials
  • ESC or EB RNA, quality assessed (see protocol 2)
  • Ambion MessageAmp II biotin‐enhanced kit (cat. no. 1791) containing:
    • T7 oligo(dT) primer
    • Arrayscript reverse transcription enzyme
    • RNAase inhibitor
    • 10× first‐strand buffer
    • dNTP mixture
    • 10× second‐strand buffer
    • DNA polymerase
    • RNAase H
    • T7 enzyme mixture
    • T7 10× reaction buffer
    • Biotin‐UTP mixture (which also includes unmodified NTPs)
    • Distilled water
  • Ambion MessageAmp II kit components (Optional; cat. no. 1751). Same as above except different in vitro transcription reagents, and random hexamer (day 2 reagent) as noted below:
    • T7 enzyme mixture
    • T7 10× reaction buffer
    • 75 mM T7 ATP solution
    • 75 mM T7 CTP solution
    • 75 mM T7 GTP solution
    • 75 mM T7 UTP solution
    • Random hexamer primer
    • Distilled water
    • Wash buffer (100% ethanol must be added before use)
    • cDNA binding buffer
    • aRNA binding buffer
    • aRNA filter cartridges
    • aRNA 1.7‐ml collection tubes
    • cDNA filter cartridges
    • cDNA 1.7‐ml collection tubes
    • 5× fragmentation buffer
  • 100% ethanol, ACS‐grade
  • 1 M Tris‐acetate, pH 8.1 (Trizma base, Sigma cat. no. T1503, pH adjusted with glacial acetic acid, Sigma cat. no. A628)
  • Magnesium acetate (Sigma cat. no. M2545)
  • Potassium acetate (Sigma cat. no. P5708)
  • DEPC‐treated water
  • Nuclease‐free 0.5‐ml tubes, non‐stick, sterile
  • Vortex
  • Microcentrifuge
  • Thermal cycler (with bonnet), incubator, or temperature block with adjustable temperatures
  • Vacuum centrifuge
  • Vacuum dryer
  • Spectrophotometer
  • Agilent Bioanalyzer or gel electrophoresis equipment, optional
  • 0.2‐µm vacuum filter unit

Support Protocol 1: Assessment of ESC and EB RNA Quality Using the Agilent Bioanalyzer

  Materials
  • 1 µg RNA isolated from ∼106 ESC and EB cells
  • Agilent Nanochip, Lab‐on‐a chip (cat. no. 5067‐1511) including:
    • Detection chips
    • Cleaning chips
    • Syringe
    • Gel matrix
    • RNA ladder
    • Spin filters
    • Gel dye concentrate
    • NanoMarker
  • Nuclease‐free water
  • Low TE buffer (10 mM Tris⋅Cl, pH 7.4 and 1 mM EDTA)
  • RNaseZap (Ambion cat. no. 9780)
  • Agilent Bioanalyzer 2100 consisting of:
    • Priming station
    • Bioanalyzer vortexer
  • 70°C heating block
  • Centrifuge
  • 1.5‐ml nuclease‐free tubes

Alternate Protocol 1: Two‐Cycle aRNA Amplification

  • cDNA (see protocol 1, steps 1 to 24)
  • Ambion MessageAmp II (cat. no. 1751) or Ambion MessageAmp II biotin‐enhanced (cat. no. 1791)
  • Random hexamer

Basic Protocol 2: Hybridizing, Washing, Staining, and Scanning of the Genechip

  Materials
  • Genechip eukaryotic hybridization control kit (Affymetrix cat. no. 900299; prokaryotic control samples used for eukaryotic samples)
  • Fragmented ES or EB aRNA (see protocol 1 or protocol 3)
  • Herring sperm DNA (Invitrogen Life Technologies cat. no. 1563‐4017)
  • 50 mg/ml acetylated bovine serum albumin (BSA; Invitrogen Life Technologies cat. no. 15561‐020)
  • 2× hybridization buffer (Quality Biological cat. no. 351‐289‐061 or see recipe)
  • Nonstringent wash buffer (Quality Biological cat. no. 351‐287‐131 or see recipe)
  • 2× MES stain buffer (Quality Biological cat. no. 351‐288‐101 or see recipe)
  • Streptavidin, R‐phycoerythrin streptavidin conjugate (SAPE; Invitrogen Life Technologies cat. no. S‐866)
  • Goat anti‐streptavidin antibody (Vector Laboratories, cat. no. BA‐0500), biotinylated
  • Goat IgG (Sigma‐Aldrich, cat. no. I 5256), reagent‐grade
  • Stringent wash buffer (Quality Biological cat. no. 351‐286‐131 or see recipe)
  • 45° and 95°C heating blocks
  • Affymetrix Genechip hybridization rotating oven
  • Affymetrix Fluidics 450 wash and stain station
  • 1.5‐ to 1.7‐ml nuclease‐free tubes
  • Affymetrix Genechip scanner 3000
  • Genechip operating system software (GCOS)

Basic Protocol 3: Data Analysis Using GCOS Batch Analysis Tool

  Materials
  • Affymetrix GCOS microarray platform
  • GCOS software (available free of charge with the purchase of the Affymetrix microarray platform)

Alternate Protocol 2: Data Analysis Using Modified PM‐Only Method

  Materials
  • First‐strand cDNA (see protocol 1, steps 1 to 9)
  • RNase H (10 U/µl; Ambion cat. no. AM12292)
  • Human glucoronidase (GUS) predeveloped control primers (Applied Biosystems cat. no. 4326320E)
  • Q‐PCR primers (Integrated DNA Technologies)
  • 2× AmpliGold PCR master mix (Applied Biosystems cat. no. E08415)
  • 1% (w/v) agarose gel, optional
  • SYBR Green PCR master mix (Applied Biosystems cat. no. 4309155)
  • 37°C water bath
  • Applied Biosystems 7900HT real‐time PCR system
  • BioAnalyzer DNA Lab‐on‐a‐chip (Agilent cat. no. 5067‐1504), optional
  • Optical adhesive covers (Applied Biosystems cat. no. 4311971)
  • Centrifuge (e.g., Eppendorf 5804)
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Figures

Videos

Literature Cited

Literature Cited
   Abeyta, M.J., Clark, A.T., Rodriguez, R.T., Bodnar, M.S., Pera, R.A., and Firpo, M.T. 2004. Unique gene expression signatures of independently derived human embryonic stem cell lines. Hum. Mol. Genet. 13:601‐608.
   Ball, C.A., Awad, I.A., Demeter, J., Gollub, J., Hebert, J.M., Hernandez‐Boussard, T., Jin, H., Matese, J.C., Nitzberg, M., Wymore, F., Zachariah, Z.K., Brown, P.O., and Sherlock, G. 2005. The Stanford Microarray Database accommodates additional microarray platforms and data formats. Nucleic Acids Res. 33:D580‐D582.
   Barrett, J.C. and Kawasaki, E.S. 2003. Microarrays: The use of oligonucleotides and cDNA for the analysis of gene expression. Drug Discov. Today 8:134‐141.
   Bhattacharya, B., Miura, T., Brandenberger, R., Mejido, J., Luo, Y., Yang, A.X., Joshi, B.H., Ginis, I., Thies, R.S., Amit, M., Lyons, I., Condie, B.G., Itskovitz‐Eldor, J., Rao, M.S., and Puri, R.K. 2004. Gene expression in human embryonic stem cell lines: Unique molecular signature. Blood 103:2956‐2964.
   Bhattacharya, B., Cai, J., Luo, Y., Miura, T., Mejido, J., Brimble, S.N., Zeng, X., Schulz, T.C., Rao, M.S., and Puri, R.K. 2005. Comparison of the gene expression profile of undifferentiated human embryonic stem cell lines and differentiating embryoid bodies. BMC Dev. Biol. 5:22.
   Brandenberger, R., Khrebtukova, I., Thies, R.S., Miura, T., Jingli, C., Puri, R., Vasicek, T., Lebkowski, J., and Rao, M. 2004. MPSS profiling of human embryonic stem cells. BMC Dev. Biol. 4:10.
   Brazma, A., Hingamp, P., Quackenbush, J., Sherlock, G., Spellman, P., Stoeckert, C., Aach, J., Ansorge, W., Ball, C.A., Causton, H.C., Gaasterland, T., Glenisson, P., Holstege, F.C., Kim, I.F., Markowitz, V., Matese, J.C., Parkinson, H., Robinson, A., Sarkans, U., Schulze‐Kremer, S., Stewart, J., Taylor, R., Vilo, J., and Vingron, M. 2001. Minimum information about a microarray experiment (MIAME)‐toward standards for microarray data. Nat. Genet. 29:365‐371.
   Brenner, S., Johnson, M., Bridgham, J., Golda, G., Lloyd, D.H., Johnson, D., Luo, S., McCurdy, S., Foy, M., Ewan, M., Roth, R., George, D., Eletr, S., Albrecht, G., Vermaas, E., Williams, S.R., Moon, K., Burcham, T., Pallas, M., DuBridge, R.B., Kirchner, J., Fearon, K., Mao, J., and Corcoran, K. 2000. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat. Biotechnol. 18:630‐634.
   Cai, J., Chen, J., Liu, Y., Miura, T., Luo, Y., Loring, J.F., Freed, W.J., Rao, M.S., and Zeng, X. 2006. Assessing self‐renewal and differentiation in human embryonic stem cell lines. Stem Cells 24:516‐530.
   Canales, R.D., Luo, Y., Willey, J.C., Austermiller, B., Barbacioru, C.C., Boysen, C., Hunkapiller, K., Jensen, R.V., Knight, C.R., Lee, K.Y., Ma, Y., Maqsodi, B., Papallo, A., Peters, E.H., Poulter, K., Ruppel, P.L., Samaha, R.R., Shi, L., Yang, W., Zhang, L., and Goodsaid, F.M. 2006. Evaluation of DNA microarray results with quantitative gene expression platforms. Nat. Biotechnol. 24:1115‐1122.
   de Reynies, A., Geromin, D., Cayuela, J.M., Petel, F., Dessen, P., Sigaux, F., and Rickman, D.S., 2006. Comparison of the latest commercial short and long oligonucleotide microarray technologies. BMC Genomics 7:51.
   Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. 1985. The in vitro development of blastocyst‐derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87:27‐45.
   Dontu, G., Abdallah, W.M., Foley, J.M., Jackson, K.W., Clarke, M.F., Kawamura, M.J., and Wicha, M.S. 2003a. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 17:1253‐1270.
   Dontu, G., Al‐Hajj, M., Abdallah, W.M., Clarke, M.F., and Wicha, M.S. 2003b. Stem cells in normal breast development and breast cancer. Cell Prolif. 36:59‐72.
   Dontu, G., Liu, S., and Wicha, M.S. 2005. Stem cells in mammary development and carcinogenesis: Implications for prevention and treatment. Stem Cell Rev. 1:207‐213.
   Fodor, S.P., Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T., and Solas, D. 1991. Light‐directed, spatially addressable parallel chemical synthesis. Science 251:767‐773.
   Ginsberg, S.D., Che, S., Counts, S.E., and Mufson, E.J. 2006. Single cell gene expression profiling in Alzheimer's disease. NeuroRx, 3:302‐318.
   Grabel, L., Becker, S., Lock, L., Maye, P., and Zanders, T. 1998. Using EC and ES cell culture to study early development: recent observations on Indian hedgehog and Bmps. Int. J. Dev. Biol. 42:917‐925.
   Hartmann, C.H. and Klein, C.A. 2006. Gene expression profiling of single cells on large‐scale oligonucleotide arrays. Nucleic Acids Res. 34:e143.
   Heins, N., Englund, M.C., Sjoblom, C., Dahl, U., Tonning, A., Bergh, C., Lindahl, A., Hanson, C., and Semb, H. 2004. Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 22:367‐376.
   Hoaglin, D.C., Mosteller, F., and Tukey, J.W. 2000. Understanding Robust and Exploratory Data Analysis. John Wiley and Sons New York.
   Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U., and Nordheim, A. 1999. Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J. Biol. Chem. 274:19838‐19845.
   Hughes, T.R., Mao, M., Jones, A.R., Burchard, J., Marton, M.J., Shannon, K.W., Lefkowitz, S.M., Ziman, M., Schelter, J.M., Meyer, M.R., Kobayashi, S., Davis, C., Dai, H., He, Y.D., Stephaniants, S.B., Cavet, G., Walker, W.L., West, A., Coffey, E., Shoemaker, D.D., Stoughton, R., Blanchard, A.P., Friend, S.H., and Linsley, P.S. 2001. Expression profiling using microarrays fabricated by an ink‐jet oligonucleotide synthesizer. Nat. Biotechnol. 19:342‐347.
   Irizarry, R.A., Hobbs, B., Collin, F., Beazer‐Barclay, Y.D., Antonellis, K.J., Scherf, U., and Speed, T.P. 2003. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249‐264.
   Itskovitz‐Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. 2000. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6:88‐95.
   Jarvinen, A.K., Hautaniemi, S., Edgren, H., Auvinen, P., Saarela, J., Kallioniemi, O.P., and Monni, O. 2004. Are data from different gene expression microarray platforms comparable? Genomics 83:1164‐1168.
   Kelly, D.L. and Rizzino, A. 2000. DNA microarray analyses of genes regulated during the differentiation of embryonic stem cells. Mol. Reprod. Dev. 56:113‐123.
   Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W., Funke, R., Gage, D., Harris, K., Heaford, A., Howland, J., Kann, L., Lehoczky, J., LeVine, R., McEwan, P., McKernan, K., Meldrim, J., Mesirov, J.P., Miranda, C., Morris, W., Naylor, J., Raymond, C., Rosetti, M., Santos, R., Sheridan, A., Sougnez, C., Stange‐Thomann, N., Stojanovic, N., Subramanian, A., Wyman, D., Rogers, J., Sulston, J., Ainscough, R., Beck, S., Bentley, D., Burton, J., Clee, C., Carter, N., Coulson, A., Deadman, R., Deloukas, P., Dunham, A., Dunham, I., Durbin, R., French, L., Grafham, D., Gregory, S., Hubbard, T., Humphray, S., Hunt, A., Jones, M., Lloyd, C., McMurray, A., Matthews, L., Mercer, S., Milne, S., Mullikin, J.C., Mungall, A., Plumb, R., Ross, M., Shownkeen, R., Sims, S., Waterston, R.H., Wilson, R.K., Hillier, L.W., McPherson, J.D., Marra, M.A., Mardis, E.R., Fulton, L.A., Chinwalla, A.T., Pepin, K.H., Gish, W.R., Chissoe, S.L., Wendl, M.C., Delehaunty, K.D., Miner, T.L., Delehaunty, A., Kramer, J.B., Cook, L.L., Fulton, R.S., Johnson, D.L., Minx, P.J., Clifton, S.W., Hawkins, T., Branscomb, E., Predki, P., Richardson, P., Wenning, S., Slezak, T., Doggett, N., Cheng, J.F., Olsen, A., Lucas, S., Elkin, C., Uberbacher, E., Frazier, M., Gibbs, R.A., Muzny, D.M., Scherer, S.E., Bouck, J.B., Sodergren, E.J., Worley, K.C., Rives, C.M., Gorrell, J.H., Metzker, M.L., Naylor, S.L., Kucherlapati, R.S., Nelson, D.L., Weinstock, G.M., Sakaki, Y., Fujiyama, A., Hattori, M., Yada, T., Toyoda, A., Itoh, T., Kawagoe, C., Watanabe, H., Totoki, Y., Taylor, T., Weissenbach, J., Heilig, R., Saurin, W., Artiguenave, F., Brottier, P., Bruls, T., Pelletier, E., Robert, C., Wincker, P., Smith, D.R., Doucette‐Stamm, L., Rubenfield, M., Weinstock, K., Lee, H.M., Dubois, J., Rosenthal, A., Platzer, M., Nyakatura, G., Taudien, S., Rump, A., Yang, H., Yu, J., Wang, J., Huang, G., Gu, J., Hood, L., Rowen, L., Madan, A., Qin, S., Davis, R.W., Federspiel, N.A., Abola, A.P., Proctor, M.J., Myers, R.M., Schmutz, J., Dickson, M., Grimwood, J., Cox, D.R., Olson, M.V., Kaul, R., Raymond, C., Shimizu, N., Kawasaki, K., Minoshima, S., Evans, G.A., Athanasiou, M., Schultz, R., Roe, B.A., Chen, F., Pan, H., Ramser, J., Lehrach, H., Reinhardt, R., McCombie, W.R., de la Bastide, M., Dedhia, N., Blocker, H., Hornischer, K., Nordsiek, G., Agarwala, R., Aravind, L., Bailey, J.A., Bateman, A., Batzoglou, S., Birney, E., Bork, P., Brown, D.G., Burge, C.B., Cerutti, L., Chen, H.C., Church, D., Clamp, M., Copley, R.R., Doerks, T., Eddy, S.R., Eichler, E.E., Furey, T.S., Galagan, J., Gilbert, J.G., Harmon, C., Hayashizaki, Y., Haussler, D., Hermjakob, H., Hokamp, K., Jang, W., Johnson, L.S., Jones, T.A., Kasif, S., Kaspryzk, A., Kennedy, S., Kent, W.J., Kitts, P., Koonin, E.V., Korf, I., Kulp, D., Lancet, D., Lowe, T.M., McLysaght, A., Mikkelsen, T., Moran, J.V., Mulder, N., Pollara, V.J., Ponting, C.P., Schuler, G., Schultz, J., Slater, G., Smit, A.F., Stupka, E., Szustakowski, J., Thierry‐Mieg, D., Thierry‐Mieg, J., Wagner, L., Wallis, J., Wheeler, R., Williams, A., Wolf, Y.I., Wolfe, K.H., Yang, S.P., Yeh, R.F., Collins, F., Guyer, M.S., Peterson, J., Felsenfeld, A., Wetterstrand, K.A., Patrinos, A., Morgan, M.J., de Jong, P., Catanese, J.J., Osoegawa, K., Shizuya, H., Choi, S., and Chen, Y.J. 2001. International Human Genome Sequencing Consortium. 2001. Initial sequencing and analysis of the human genome. Nature 409:860‐921.
   Li, C. and Wong, W.H. 2001. Model‐based analysis of oligonucleotide arrays: Expression index computation and outlier detection. Proc. Natl. Acad. Sci. U.S.A. 98:31‐36.
   Li, J., Spletter, M.L., and Johnson, J.A. 2005. Dissecting tBHQ‐induced ARE‐driven gene expression through long and short oligonucleotide arrays. Physiol. Genomics 21:43‐58.
   Liu, Y., Shin, S., Zeng, X., Zhan, M., Gonzalez, R., Mueller, F.J., Schwartz, C.M., Xue, H., Li, H., Baker, S.C., Chudin, E., Barker, D.L., McDaniel, T.K., Oeser, S., Loring, J.F., Mattson, M.P., and Rao, M.S. 2006. Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines. BMC Dev. Biol. 6:20.
   Loring, J.F. and Rao, M.S. 2006. Establishing standards for the characterization of human embryonic stem cell lines. Stem Cells 24:145‐150.
   Mecham, B.H., Klus, G.T., Strovel, J., Augustus, M., Byrne, D., Bozso, P., Wetmore, D.Z., Mariani, T.J., Kohane, I.S., and Szallasi, Z. 2004. Sequence‐matched probes produce increased cross‐platform consistency and more reproducible biological results in microarray‐based gene expression measurements. Nucleic Acids Res. 32:e74.
   Miller, R.M. and Federoff, H.J. 2006. Microarrays in Parkinson's disease: A systematic approach. NeuroRx 3:319‐326.
   Mitalipova, M., Calhoun, J., Shin, S., Wininger, D., Schulz, T., Noggle, S., Venable, A., Lyons, I., Robins, A., and Stice, S. 2003. Human embryonic stem cell lines derived from discarded embryos. Stem Cells 21:521‐526.
   Miura, T., Luo, Y., Khrebtukova, I., Brandenberger, R., Zhou, D., Thies, R.S., Vasicek, T., Young, H., Lebkowski, J., Carpenter, M.K., and Rao, M.S. 2004. Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan. Stem Cells Dev. 13:694‐715.
   Novoradovskaya, N., Whitfield, M.L., Basehore, L.S., Novoradovsky, A., Pesich, R., Usary, J., Karaca, M., Wong, W.K., Aprelikova, O., Fero, M., Perou, C.M., Botstein, D., and Braman, J. 2004. Universal Reference RNA as a standard for microarray experiments. BMC Genomics 5:20.
   Ohlsson, C., Lovstedt, K., Holmes, P.V., Nilsson, A., Carlsson, L., and Tornell, J. 1993. Embryonic stem cells express growth hormone receptors: Regulation by retinoic acid. Endocrinology 133:2897‐2903.
   Peano, C., Severgnini, M., Cifola, I., De Bellis, G., and Battaglia, C. 2006. Transcriptome amplification methods in gene expression profiling. Expert Rev. Mol. Diagn. 6:465‐480.
   Player, A., Wang, Y., Bhattacharya, B., Rao, M., Puri, R.K., and Kawasaki, E.S. 2006. Comparisons between transcriptional regulation and RNA expression in human embryonic stem cell lines. Stem Cells Dev. 15:315‐323.
   Reinartz, J., Bruyns, E., Lin, J.Z., Burcham, T., Brenner, S., Bowen, B., Kramer, M., and Woychik, R. 2002. Massively parallel signature sequencing (MPSS) as a tool for in‐depth quantitative gene expression profiling in all organisms. Brief Funct. Genomic Proteomic 1:95‐104.
   Richards, M., Tan, S.P., Tan, J.H., Chan, W.K., and Bongso, A. 2004. The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22:51‐64.
   Richards, M., Tan, S.P., Chan, W.K., Bongso, A., Richards, M., Tan, S.P., Tan, J.H., Chan, W.K., and Bongso, A. 2006. Reverse serial analysis of gene expression (SAGE) characterization of orphan SAGE tags from human embryonic stem cells identifies the presence of novel transcripts and antisense transcription of key pluripotency genes. The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 24:1162‐1173.
   Robson, P. 2004. The maturing of the human embryonic stem cell transcriptome profile. Trends Biotechnol. 22:609‐612.
   Sato, N., Sanjuan, I.M., Heke, M., Uchida, M., Naef, F., and Brivanlou, A.H. 2003. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev. Biol. 260:404‐413.
   Sell, S. 2001. The role of progenitor cells in repair of liver injury and in liver transplantation. Wound Repair Regen. 9:467‐482.
   Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P.J., Blumenthal, P.D., Huggins, G.R., and Gearhart, J.D. 1998. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. U.S.A. 95:13726‐13731.
   Shamblott, M.J., Axelman, J., Littlefield, J.W., Blumenthal, P.D., Huggins, G.R., Cui, Y., Cheng, L., and Gearhart, J.D. 2001. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc. Natl. Acad. Sci. U.S.A. 98:113‐118.
   Shi, L., Tong, W., Fang, H., Scherf, U., Han, J., Puri, R.K., Frueh, F.W., Goodsaid, F.M., Guo, L., Su, Z., Han, T., Fuscoe, J.C., Xu, Z.A., Patterson, T.A., Hong, H., Xie, Q., Perkins, R.G., Chen, J.J., and Casciano, D.A. 2005. Cross‐platform comparability of microarray technology: Intra‐platform consistency and appropriate data analysis procedures are essential. BMC Bioinformatics 6:S12.
   Shi, L., Reid, L.H., Jones, W.D., Shippy, R., Warrington, J.A., Baker, S.C., Collins, P.J., de Longueville, F., Kawasaki, E.S., Lee, K.Y., Luo, Y., Sun, Y.A., Willey, J.C., Setterquist, R.A., Fischer, G.M., Tong, W., Dragan, Y.P., Dix, D.J., Frueh, F.W., Goodsaid, F.M., Herman, D., Jensen, R.V., Johnson, C.D., Lobenhofer, E.K., Puri, R.K., Schrf, U., Thierry‐Mieg, J., Wang, C., Wilson, M., Wolber, P.K., Zhang, L., Amur, S., Bao, W., Barbacioru, C.C., Lucas, A.B., Bertholet, V., Boysen, C., Bromley, B., Brown, D., Brunner, A., Canales, R., Cao, X.M., Cebula, T.A., Chen, J.J., Cheng, J., Chu, T.M., Chudin, E., Corson, J., Corton, J.C., Croner, L.J., Davies, C., Davison, T.S., Delenstarr, G., Deng, X., Dorris, D., Eklund, A.C., Fan, X.H., Fang, H., Fulmer‐Smentek, S., Fuscoe, J.C., Gallagher, K., Ge, W., Guo, L., Guo, X., Hager, J., Haje, P.K., Han, J., Han, T., Harbottle, H.C., Harris, S.C., Hatchwell, E., Hauser, C.A., Hester, S., Hong, H., Hurban, P., Jackson, S.A., Ji, H., Knight, C.R., Kuo, W.P., LeClerc, J.E., Levy, S., Li, Q.Z., Liu, C., Liu, Y., Lombardi, M.J., Ma, Y., Magnuson, S.R., Maqsodi, B., McDaniel, T., Mei, N., Myklebost, O., Ning, B., Novoradovskaya, N., Orr, M.S., Osborn, T.W., Papallo, A., Patterson, T.A., Perkins, R.G., Peters, E.H., Peterson, R., Philips, K.L., Pine, P.S., Pusztai, L., Qian, F., Ren, H., Rosen, M., Rosenzweig, B.A., Samaha, R.R., Schena, M., Schroth, G.P., Shchegrova, S., Smith, D.D., Staedtler, F., Su, Z., Sun, H., Szallasi, Z., Tezak, Z., Thierry‐Mieg, D., Thompson, K.L., Tikhonova, I., Turpaz, Y., Vallanat, B., Van, C., Walker, S.J., Wang, S.J., Wang, Y., Wolfinger, R., Wong, A., Wu, J., Xiao, C., Xie, Q., Xu, J., Yang, W., Zhang, L., Zhong, S., Zong, Y., and Slikker, W. 2006. The MicroArray Quality Control (MAQC) project shows inter‐ and intraplatform reproducibility of gene expression measurements. Nat. Biotechnol. 24:1151‐1161.
   Shin, S. and Rao, M.S. 2006. Large‐scale analysis of neural stem cells and progenitor cells. Neurodegener Dis. 3:106‐111.
   Situma, C., Hashimoto, M., and Soper, S.A. 2006. Merging microfluidics with microarray‐based bioassays. Biomol. Eng. 23:213‐231.
   Skottman, H., Mikkola, M., Lundin, K., Olsson, C., Stromberg, A.M., Tuuri, T., Otonkoski, T., Hovatta, O., and Lahesmaa, R. 2005. Gene expression signatures of seven individual human embryonic stem cell lines. Stem Cells 23:1343‐1356.
   Suhr, S.T. and Gage, F.H. 1993. Gene therapy for neurologic disease. Arch. Neurol. 50:1252‐1268.
   Suhr, S.T. and Gage, F.H. 1999. Gene therapy in the central nervous system: The use of recombinant retroviruses. Arch. Neurol. 56:287‐292.
   Takahashi, K. and Yamanaka, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663‐676.
   Tatematsu, M., Tsukamoto, T., and Inada, K. 2003. Stem cells and gastric cancer: Role of gastric and intestinal mixed intestinal metaplasia. Cancer Sci. 94:135‐141.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145‐1147.
   Tietjen, I., Rihel, J.M., Cao, Y., Koentges, G., Zakhary, L., and Dulac, C. 2003. Single‐cell transcriptional analysis of neuronal progenitors. Neuron 38:161‐175.
   Tong, W., Lucas, A.B., Shippy, R., Fan, X., Fang, H., Hong, H., Orr, M.S., Chu, T.M., Guo, X., Collins, P.J., Sun, Y.A., Wang, S.J., Bao, W., Wolfinger, R.D., Shchegrova, S., Guo, L., Warrington, J.A., and Shi, L. 2006. Evaluation of external RNA controls for the assessment of microarray performance. Nat. Biotechnol. 24:1132‐1139.
   Tsuji, I., Mitani, T., Mitsuhashi, A., Watanabe, Y., Hosoi, Y., and Hoshiai, H. 2006. Inhibition of Oct4 expression in mouse preimplantation embryos using morpholino antisense oligonucleotides. Tohoku J. Exp. Med. 208:333‐342.
   Wei, C.L., Miura, T., Robson, P., Lim, S.K., Xu, X.Q., Lee, M.Y., Gupta, S., Stanton, L., Luo, Y., Schmitt, J., Thies, S., Wang, W., Khrebtukova, I., Zhou, D., Liu, E.T., Ruan, Y.J., Rao, M., and Lim, B. 2005. Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23:166‐185.
   Wu, Q., Chen, X., Zhang, J., Loh, Y.H., Low, T.Y., Zhang, W., Zhang, W., Sze, S.K., Lim, B., and Ng, H.H. 2006. Sall4 interacts with Nanog and co‐occupies Nanog genomic sites in embryonic stem cells. J. Biol. Chem. 281:24090‐24094.
   Yang, A.X., Mejido, J., Luo, Y., Zeng, X., Schwartz, C., Wu, T., Thies, R.S., Bhattacharya, B., Han, J., Freed, B., Rao, M., and Puri, R.K. 2005. Development of a focused microarray to assess human embryonic stem cell differentiation. Stem Cells Dev. 14:270‐284.
   Zhan, M., Miura, T., Xu, X., and Rao, M.S. 2005. Conservation and variation of gene regulation in embryonic stem cells assessed by comparative genomics. Cell Biochem. Biophys. 43:379‐405.
   Zhang, J., Tam, W.L., Tong, G.Q., Wu, Q., Chan, H.Y., Soh, B.S., Lou, Y., Yang, J., Ma, Y., Chai, L., Ng, H.H., Lufkin, T., Robson, P., and Lim, B. 2006a. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat. Cell Biol. 8:1114‐1123.
   Zhang, P., Kerkela, E., Skottman, H., Levkov, L., Kivinen, K., Lahesmaa, R., Hovatta, O., and Kere, J. 2006b. Distinct sets of developmentally regulated genes that are expressed by human oocytes and human embryonic stem cells. Fertil. Steril. 87:677‐690.
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