Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Direct in situ reverse transcriptase-polymerase chain reaction.

R Kher1, R Bacallao

  • 1Division of Nephrology and Hypertension, Richard Roudebusch Veterans Affairs Medical Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

American Journal of Physiology. Cell Physiology
|July 10, 2001
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Compromised cytoarchitecture and polarized trafficking in autosomal dominant polycystic kidney disease cells.

The Journal of cell biology·2000
Same author

Role of the matrix in autosomal dominant polycystic kidney disease.

Renal failure·1998
Same author

Optical methods in renal research.

Seminars in nephrology·1998
Same author

NHE-1 protein in vascular smooth muscle and lymphocytes from the spontaneously hypertensive rat.

Hypertension (Dallas, Tex. : 1979)·1997
Same author

Antifertility effects of an LHRH agonist in male mice.

Contraception·1996
Same author

Actin and villin compartmentation during ATP depletion and recovery in renal cultured cells.

Kidney international·1995
Same journal

A non-hydrolyzable candesartan cilexetil analog reveals synergistic activation as a tractable mechanism for TMEM175 modulation.

American journal of physiology. Cell physiology·2026
Same journal

Mitochondrial Calcium Transport in Amino Acid Metabolism: From nutritional responses to metabolic regulation.

American journal of physiology. Cell physiology·2026
Same journal

N-linked glycosylation regulates SNAT2 trafficking and stability in pancreatic ductal adenocarcinoma cells.

American journal of physiology. Cell physiology·2026
Same journal

Oxaloacetate Inhibition of Succinate Dehydrogenase: Mechanism and Physiological Implications.

American journal of physiology. Cell physiology·2026
Same journal

miR-339-5p impairs myogenic proliferation and differentiation by repressing contractile gene programs in skeletal muscle.

American journal of physiology. Cell physiology·2026
Same journal

Impact of ΔF508 CFTR Mutation on Diaphragm Function During Acute Inflammation.

American journal of physiology. Cell physiology·2026
See all related articles

This study introduces a novel method for in situ reverse transcriptase-polymerase chain reaction (RT-PCR) to accurately detect low-abundance mRNA in cells. By eliminating nonspecific amplification, this technique enhances specificity and enables rapid cellular mRNA detection for potential pathological diagnosis.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • In situ hybridization offers cellular localization of nucleic acids but has low sensitivity.
  • In situ reverse transcriptase-polymerase chain reaction (RT-PCR) improves sensitivity for detecting low-abundance mRNA.
  • Existing in situ RT-PCR methods suffer from nonspecific amplification due to mispriming or residual genomic DNA.

Purpose of the Study:

  • To develop a more specific and sensitive method for in situ detection of cellular mRNA.
  • To overcome limitations of nonspecific amplification in current in situ RT-PCR techniques.
  • To enable rapid and accurate identification of target mRNA within cells.

Main Methods:

  • Pretreatment of samples with restriction enzymes before DNase I digestion to eliminate background amplification.

Related Experiment Videos

  • Utilizing primers tagged with a far-red shifted fluorescent dye (e.g., Cy5) for PCR reactions.
  • Detection of target mRNA via fluorescence microscopy.

    • Main Results:

    • Nonspecific background amplification was successfully eliminated by the novel pretreatment protocol.
    • The modified method demonstrated increased specificity in cellular mRNA detection.
    • Rapid and accurate in situ identification of target mRNA was achieved.

    Conclusions:

    • The developed modifications significantly enhance the specificity and speed of in situ mRNA detection.
    • This improved technique effectively addresses issues of nonspecific amplification in cellular mRNA analysis.
    • The method holds potential for application in pathological diagnosis due to its accuracy and efficiency.