Using Host 28S Ribosomal RNA as a Housekeeping Gene for Quantitative Real‐Time Reverse Transcription‐PCR (qRT‐PCR) in Virus‐Infected Animal Cells

Jian‐Li Xue1, Xiao‐Wen Cheng1

1 Miami University, Oxford, Ohio
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1D.2
DOI:  10.1002/9780471729259.mc01d02s19
Online Posting Date:  November, 2010
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Abstract

The use of quantitative real‐time reverse transcription‐PCR (qRT‐PCR) for studying regulation of gene transcription requires an internal template‐loading control or a housekeeping gene to guarantee the validity of the data collection, analysis, and interpretation. Analysis of gene transcription in virus‐infected animal cells is problematic because virus infection often results in modified or fluctuating gene transcription patterns of conventionally used housekeeping genes, such as the glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) gene and the β‐actin gene. It has been demonstrated that the host 28S ribosomal gene can be used as a housekeeping gene in qRT‐PCR in virus‐infected insect cells. The stability of the human 28S rRNA gene transcription during the infection of HeLa cells with adenovirus has been confirmed, and this method has been extended to the use of the human 28S rRNA gene as a housekeeping gene in adenovirus‐infected HeLa cells. Step‐by‐step instructions are described for use of this control in analysis of gene transcription in both types of virus‐infected animal cells. Curr. Protoc. Microbiol. 19:1D.2.1‐1D.2.13. © 2010 by John Wiley & Sons, Inc.

Keywords: qRT‐PCR; housekeeping gene; 28S rRNA; HeLa cells; insect cells; baculovirus; adenovirus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Virus Infection of Animal Cells and Sample Harvest
  • Basic Protocol 2: Extraction of RNA from Virus‐Infected Animal Cells
  • Basic Protocol 3: Quality Assessment of Extracted RNA by Denaturing Agarose Gel Electrophoresis
  • Basic Protocol 4: Removal of Genomic DNA Contamination from RNA Samples
  • Basic Protocol 5: cDNA Synthesis
  • Basic Protocol 6: Amplification of cDNA by a Bio‐Rad iCycler iQ System
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Virus Infection of Animal Cells and Sample Harvest

  Materials
  • Cell lines: IPLB‐Sf21‐AE (Sf21) cells for baculovirus (Autographa californicamulticapsid nucleopolyhedrovirus; AcMNPV) or HeLa cells for adenovirus (Adenovirus type 5 or Ad5)
  • Cell culture media: TNM‐FH medium supplemented with 10% fetal bovine serum (FBS) and antibiotics penicillin (100 U/ml) and streptomycin (100 µg/ml) for Sf21 or Dulbecco's Modification of Eagle's medium supplemented with 10% FBS and antibiotics penicillin (100 U/ml) and streptomycin (100 µg/ml) for HeLa cells (see O'Reilly et al., and unit 14.1)
  • Virus stock with known titers
  • Liquid nitrogen
  • Hemacytometer
  • 6‐well tissue culture plates
  • Cell culture incubator (27°C) for insect cells or CO 2 incubator (37°C)
  • 27°C rocking incubator
  • Pasteur pipets
  • 15‐ml centrifuge tubes
  • Benchtop centrifuges

Basic Protocol 2: Extraction of RNA from Virus‐Infected Animal Cells

  Materials
  • Ice
  • Guanidinium thiocyanate
  • 1 M sodium citrate pH 7.0
  • N‐lauroylsarcosine (sarkosyl)
  • 2‐mercaptoethanol (2‐ME)
  • Frozen or fresh samples (see protocol 1)
  • 2 M sodium acetate pH 4.2
  • DEPC‐treated water saturated phenol
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 2‐propanol (isopropanol)
  • 100% ethanol
  • 70% ethanol (diluted in sterile, DEPC‐treated water)
  • Sterile, DEPC‐treated H 2O
  • Gloves
  • Ice buckets
  • 1‐ml, 200‐µl, and 10‐µl micropipettors and corresponding RNase‐free tips
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge tube racks
  • Refrigerated microcentrifuge, 4°C
  • NanoDrop Spectrophotometer (Thermo Scientific), or equivalent
CAUTION: DEPC is a suspected carcinogen and it should be handled with great care. Wear gloves and use a fume hood when using this chemical.

Basic Protocol 3: Quality Assessment of Extracted RNA by Denaturing Agarose Gel Electrophoresis

  Materials
  • Agarose
  • 10 × MOPS running buffer (see recipe)
  • 37% formaldehyde (12.3 M)
  • Frozen RNA samples (see protocol 2)
  • DEPC‐treated H 2O
  • Formamide
  • 10 mg/ml ethidium bromide staining solution (Fisher Scientific)
  • Formaldehyde loading buffer (see recipe)
  • Agarose gel box, tray, comb, casting apparatus
  • Sterile, RNase‐free flask
  • 1.5‐ml RNase‐free microcentrifuge tubes, sterile
  • Gel electrophoresis power supply
  • UV light box

Basic Protocol 4: Removal of Genomic DNA Contamination from RNA Samples

  Materials
  • Extracted RNA from virus‐infected animal cells
  • 10× DNase buffer (Promega)
  • RQ1 RNase‐Free DNase (Promega)
  • DEPC‐treated H 2O, sterile
  • Stop buffer (Promega)
  • 2 M sodium acetate, pH 4.2
  • 100% ethanol
  • 70% ethanol (diluted in sterile, DEPC‐treated water)
  • 1.5‐ml RNase‐free microcentrifuge tubes, sterile
  • 37° and 65°C water baths
  • Centrifuge

Basic Protocol 5: cDNA Synthesis

  Materials
  • DyNAmo cDNA synthesis kit (New England Biolabs; or equivalents) containing:
    • M‐MuLV RNase H+ Reverse Transcriptase
    • RT reaction buffer (2×)
    • Oligo (dT) 15 primer (100 ng/µl)
    • Random hexamers (300 ng/µl)
  • 28S rRNA reverse primer (20 µM) (Table 1.2.2; Xue et al., )
  • DNase‐treated RNA ( protocol 4)
  • RNase‐free, sterile, thin‐walled PCR tubes
  • Thermal cycler
    Table 1.0.2   MaterialsSequences of 28S rRNA Primers for Insect Sf21 Cells and Human Cells

    Primer name Sequences
    28S‐F (insect Sf21 cell ) 5′‐CGA CGT TGC TTT TTG ATC CT‐3′
    28S‐R (insect Sf21 cell ) 5′‐GCA ACG ACA AGC CAT CAG TA‐3′
    28S‐F (human cell) 5′‐AAC GAG ATT CCC ACT GTC CC‐3′
    28S‐R (human cell) 5′‐CTT CAC CGT GCC AGA CTA GAG‐3′

Basic Protocol 6: Amplification of cDNA by a Bio‐Rad iCycler iQ System

  Materials
  • iQ SYBR Green supermix kit (Bio‐Rad) containing 2× supermix
  • Forward and reverse primers (Table 1.2.2)
  • DEPC‐treated water
  • Bio‐Rad iCycler iQ system
  • 1.5‐ml microcentrifuge tubes
  • 0.2‐ml PCR white 8‐tube strip with clear flat 8‐cap strips (USA Scientific)
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Figures

Videos

Literature Cited

Literature Cited
   Bustin, S.A. 2002. Quantification of mRNA using real‐time reverse transcription PCR (RT‐PCR): Trends and problems. J. Mol. Endocrinol. 29:23‐39.
   Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
   Goidin, D., Mamessier, A., Staquet, M.J., Schmitt, D., and Berthier‐Vergnes, O. 2001. Ribosomal 18S RNA prevails over glyceraldehyde‐3‐phosphate dehydrogenase and beta‐actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal. Biochem. 295:17‐21.
   Hardwick, J.M. 2000. Cyclin' on the viral path to destruction. Nat. Cell Biol 2:203‐204.
   Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M. 1996. Real time quantitative PCR. Genome Res. 6:986‐994.
   Hussain, M., Taft, R.J., and Asgari, S. 2008. An insect virus‐encoded microRNA regulates viral replication. J. Virol. 82:9164‐9170.
   Mackay, I.M., Arden, K.E., and Nitsche, A. 2002. Real‐time PCR in virology. Nucleic Acids Res. 30:1292‐1305.
   O'Reilly, D.R., Miller, L.K., and Luckow, V.A. 1992. Baculovirus expression vectors: A laboratory manual. W.H. Freeman & Co., New York.
  Pfaffl, M.W. 2001. A new mathematical model for relative quantification in real‐time RT‐PCR. Nucleic Acids Res. 29:2002‐2007.
   Radonic, A., Thulke, S., Mackay, I.M., Landt, O., Siegert, W., and Nitsche, A. 2004. Guideline to reference gene selection for quantitative real‐time PCR. Biochem. Biophys. Res. Commun. 313:856‐862.
   Radonic, A., Thulke, S., Bae, H.G., Muller, M.A., Siegert, W., and Nitsche, A. 2005. Reference gene selection for quantitative real‐time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus‐6, Camelpox virus and Cytomegalovirus infections. Virol. J. 2:7.
   Salem, T.Z., Turney, C.M., Wang, L., Xue, J.L., Wan, X.F., and Cheng, X.W. 2008. Transcriptional analysis of a major capsid protein gene from Spodoptera exigua ascovirus 5a. Arch. Virol. 153:149‐162.
   Schlesinger, J., Tonjes, M., Schueler, M., Zhang, Q., Dunkel, I., and Sperling, S.R. 2010. Evaluation of the LightCycler 1536 Instrument for high‐throughput quantative real‐time PCR. Methods 50:S19‐S22.
   Schmittgen, T.D. and Zakrajsek, B.A. 2000. Effect of experimental treatment on housekeeping gene expression: Validation by real‐time, quantitative RT‐PCR. J. Biochem. Biophys. Methods 46:69‐81.
   Xue, J.L., Salem, T.Z., Turney, C.M., and Cheng, X.W. 2010. Strategy of the use of 28S rRNA as a housekeeping gene in real‐time quantitative PCR analysis of gene transcription in insect cells infected by viruses. J. Virol. Methods 163:210‐215.
   Zhong, H. and Simons, J.W. 1999. Direct comparison of GAPDH, beta‐actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem. Biophys. Res. Commun. 259:523‐526.
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