Direct in situ reverse transcriptase-polymerase chain reaction

Research output: Contribution to journalArticle

8 Citations (Scopus)

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
JournalAmerican Journal of Physiology - Cell Physiology
Volume281
Issue number2 50-2
StatePublished - 2001

Fingerprint

Polymerase chain reaction
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Amplification
Messenger RNA
Nucleic acid sequences
Deoxyribonuclease I
Fluorescence microscopy
Fluorescent Dyes
Fluorescence Microscopy
Nucleic Acids
In Situ Hybridization
Digestion
Tissue
Polymerase Chain Reaction
DNA
Enzymes

Keywords

  • Deoxyribonuclease I
  • Restriction enzymes

ASJC Scopus subject areas

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

Cite this

Direct in situ reverse transcriptase-polymerase chain reaction. / Kher, R.; Bacallao, Robert.

In: American Journal of Physiology - Cell Physiology, Vol. 281, No. 2 50-2, 2001.

Research output: Contribution to journalArticle

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