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Related Concept Videos

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Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Direct in situ rt-PCR.

Laura Lossi1, Graziana Gambino, Chiara Salio

  • 1Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy. lossi.laura@unito.it

Methods in Molecular Biology (Clifton, N.J.)
|September 17, 2011
PubMed
Summary
This summary is machine-generated.

In situ polymerase chain reaction (PCR) enables direct detection of messenger RNA (mRNA) in tissue sections. This method combines PCR with in situ hybridization for precise mRNA localization in various biological samples.

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Area of Science:

  • Histology
  • Molecular Biology
  • Neuroscience

Background:

  • In situ hybridization is traditionally used for mRNA localization.
  • Polymerase chain reaction (PCR) offers sensitive nucleic acid detection.
  • Combining these techniques allows for direct mRNA detection within tissue architecture.

Purpose of the Study:

  • To describe the application of in situ PCR for neuropeptide mRNA localization.
  • To provide a detailed protocol for direct in situ reverse transcription PCR (RT-PCR).
  • To enable nonradioactive detection of mRNA in fixed and embedded tissues.

Main Methods:

  • Utilizes in situ polymerase chain reaction (PCR) on tissue sections.
  • Incorporates reverse transcription (RT) for RNA to cDNA conversion.
  • Employs digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) for nonradioactive detection.
  • Detection involves an anti-digoxigenin antibody conjugated with alkaline phosphatase.

Main Results:

  • Successful localization of neuropeptide mRNA directly within tissue sections.
  • A detailed protocol for performing in situ RT-PCR is presented.
  • The method allows for visualization of mRNA distribution at a cellular level.

Conclusions:

  • In situ PCR is a powerful histological technique for mRNA localization.
  • The described protocol offers a reliable method for neuropeptide mRNA detection.
  • The technique is adaptable for fluorescent probes and combination with immunocytochemistry.