Micro RT‐PCR

Cloud P. Paweletz1, Lu Charboneau1, Lance A. Liotta1

1 National Cancer Institute/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Appendix 3G
DOI:  10.1002/0471143030.cba03gs10
Online Posting Date:  May, 2001
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Abstract

This unit presents a collection of protocols for the microisolation, manipulation, and amplification of the RNA content of microdissected cells. Even though emphasis in these protocols is given for microdissected cells, these protocols have successfully been used for bulk tissue (i.e., less than 10 ug).

     
 
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Table of Contents

  • Basic Protocol 1: Micro RT‐PCR
  • Support Protocol 1: Microisolation of RNA
  • Commentary
     
 
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Materials

Basic Protocol 1: Micro RT‐PCR

  Materials
  • DEPC‐treated H 2O ( appendix 2A)
  • 20 U/µl RNase inhibitor (ABI‐Perkin Elmer)
  • RNA pellet, dry ( protocol 2)
  • 5× RT buffer (GenHunter)
  • 250 µM dNTPs (GenHunter)
  • 10 µM random‐hexamer or oligo(dT) primers (ABI‐Perkin Elmer)
  • 100 U/µl MMLV reverse transcriptase (Stratagene)
  • 10× PCR buffer (Perkin Elmer): 500 KCl/15 mM MgCl 2/1 mg/ml gelatin in 100 mM Tris⋅Cl, pH 8.4( appendix 2A)
  • 10 µM upstream primer
  • 10 µM downstream primer
  • 5 U/µl Taq DNA polymerase (ABI‐Perkin Elmer)
  • 10 mCi/ml 32P‐dCTP or 33P‐dCTP (∼300 Ci/mmol)
  • 12‐µl PCR tubes, RNase‐free
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 1.5)
CAUTION: DEPC is a suspected carcinogen and must be handled with caution.

Support Protocol 1: Microisolation of RNA

  • Tissue
  • Micro RNA Isolation kit (Stratagene):
  •  Denaturing solution
  •  2‐mercaptoethanol
  •  H 2O‐saturated phenol
  •  2 M sodium acetate, pH 4.0
  •  1 µg/µl glycogen
  •  Isopropanol, cold
  •  49:1 (v/v) chloroform/isoamyl alcohol
  • 60% ethanol/40% DEPC‐treated H 2O ( appendix 2A)
  • DEPC‐treated H 2O ( appendix 2A)
  • Message Clean kit (GenHunter):
  •  RNase inhibitor
  •  10× reaction buffer
  •  10 U/µl mg/ml DNase
  • RNase Away (Molecular Bio‐Products)
  • RNase‐free tubes (Eppendorf)
  • Additional reagents and equipment for microdissection (unit 2.5)
CAUTION: DEPC is a suspected carcinogen and must be handled with caution.NOTE: Because the starting material is only between 10 and 100 ng, it is of absolute necessity to use only DEPC‐treated water to inactivate ribonucleases, to use only labware certified “RNase free,” and to always wear gloves.
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Figures

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Literature Cited

Literature Cited
   Chirgwin, J.J., Przbyla, A.E., MacDonald, R.J., and Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294‐5299.
   Emmert‐Buck, M.R., Bonner, R.F., Smith, P.D., Chaqui, R.F., Zhuang, Z., Goldstein, S.R., Weiss, R.A., and Liotta, L.A. 1996. Laser capture microdissection. Science 274:998‐1001.
   Kirby, K.S. 1968. Isolation of nucleic acids, with phenolic solvents. Methods Enzymol. 12B:87‐98.
   Krizman, D.B., Chuaqui, R.F., Meltzer, P.S., Trent, J.M., Duray, P.H., Lineham, W.M., Liotta, L.A., and Emmert‐Buck, M.R. 1996. Construction of a representative cDNA library from prostatic intraepithelial neoplasia. Cancer Res. 56:5380‐5381.
   Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rutter, W.J., and Goodman, H.M. 1977. Rat insulin genes: Construction of plasmids containing the coding sequence. Science 196:1313‐1319.
   Veres, G., Gibbs, R.A., Scherer, S.E., and Caskey, C.T. 1987. The molecular basis of the sparse fur mouse mutation. Science 237:415‐417.
Internet Resources
  http://cgap-mf.nih.gov/protocols
  An excellent source for recovery and analysis of RNA from microdissected cells.
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