A versatile ELISA-PCR assay for mRNA quantitation from a few cells

Biotechniques. 1993 Oct;15(4):730-7.

Abstract

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step: < 0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Avidin
  • Base Sequence
  • Biotin
  • Cell Line
  • DNA, Viral / analysis
  • Digoxigenin
  • Enzyme-Linked Immunosorbent Assay*
  • HIV / genetics
  • Interleukin-2 / genetics
  • Molecular Sequence Data
  • Nucleic Acid Hybridization
  • Oligonucleotide Probes
  • Polymerase Chain Reaction*
  • RNA, Messenger / analysis*

Substances

  • DNA, Viral
  • Interleukin-2
  • Oligonucleotide Probes
  • RNA, Messenger
  • Avidin
  • Biotin
  • Digoxigenin