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

Low-ratio hybridization subtraction.

J Fargnoli1, N J Holbrook, A J Fornace

  • 1Laboratory of Molecular Genetics, NIA, NIH, Baltimore, Maryland 21224.

Analytical Biochemistry
|June 11, 1990
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

p21(Waf1) is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate.

Cell cycle (Georgetown, Tex.)·2010
Same author

Day/night variations of high-molecular-weight adiponectin and lipocalin-2 in healthy men studied under fed and fasted conditions.

Diabetologia·2010
Same author

The role of the MKK6/p38 MAPK pathway in Wip1-dependent regulation of ErbB2-driven mammary gland tumorigenesis.

Oncogene·2006
Same author

Genomic instability in Gadd45a-/- cells is coupled with S-phase checkpoint defects.

Cell cycle (Georgetown, Tex.)·2005
Same author

Gadd45a acts as a modifier locus for lymphoblastic lymphoma.

Leukemia·2005
Same author

A nucleotide excision repair master-switch: p53 regulated coordinate induction of global genomic repair genes.

Cancer biology & therapy·2002

A new low-ratio hybridization subtraction method effectively enriches for complementary DNA (cDNA) from low-abundance transcripts. This technique aids in identifying gene expression changes between cell populations.

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Identifying differentially expressed genes is crucial for understanding biological processes and disease mechanisms.
  • Traditional methods may lack sensitivity for detecting low-abundance transcripts with modest expression changes.
  • Enrichment strategies are needed to isolate specific complementary DNA (cDNA) populations for further analysis.

Purpose of the Study:

  • To develop and validate a low-ratio hybridization subtraction protocol for enriching cDNA from elevated transcripts.
  • To optimize conditions for cloning and identifying induced transcripts from enriched cDNA libraries.
  • To evaluate the impact of reverse transcriptase choice on the enrichment of long RNA transcripts.

Main Methods:

  • Development of a low-ratio hybridization subtraction protocol using RNA and cDNA.

Related Experiment Videos

  • Cloning of enriched cDNA and screening of the cDNA library using specific probes.
  • Comparative analysis of different reverse transcriptases (MMLV and AMV) for cDNA synthesis.

    • Main Results:

    • The low-ratio hybridization subtraction protocol achieved substantial enrichment of low-abundance, several-fold induced transcripts.
    • Efficient identification of clones encoding induced transcripts was possible through probe screening.
    • Moloney Murine Leukemia Virus (MMLV) reverse transcriptase facilitated enrichment of cDNA for RNA up to 5 kb, while Avian Myeloblastosis Virus (AMV) was limited to <1 kb.

    Conclusions:

    • Low-ratio hybridization subtraction is an effective method for enriching specific cDNA populations, particularly for low-abundance transcripts.
    • The choice of reverse transcriptase significantly influences the length of RNA transcripts that can be enriched.
    • This protocol provides a valuable tool for gene discovery and analyzing differential gene expression.