Direct in situ reverse transcriptase-polymerase chain reaction

R. Kher, R. Bacallao

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.

Original languageEnglish (US)
Pages (from-to)C726-C732
JournalAmerican Journal of Physiology - Cell Physiology
Volume281
Issue number2 50-2
StatePublished - Aug 27 2001

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Keywords

  • Deoxyribonuclease I
  • Restriction enzymes

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

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