A new quantitative film autoradiographic method of quantifying mRNA transcripts for in situ hybridization

Arpad Palfi, Laszlo Hatvani, Karoly Gulya

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

We developed and tested a novel quantitative method for the quantification of film autoradiographs, involving a mathematical model and a dot-blot-based membrane standard scale. The exponential model introduced here, ROD = p1(1 - exp[p2x]), appropriately (r2>0.999), describes the relation between relative optical density (ROD) and radioactivity (x) in the range between 0 and 240 gray scale values (using a 256-gray scale level digitizer). By means of this model, standard curves with distinct quenching properties can be exactly interconverted, permitting the tissue-equivalent calibration of different standard scales. The membrane standard scale employed here has several advantages, including the flexible radioactivity range, the facile and rapid preparation technique, and the compact size. The feasibility of the quantification procedure is exemplified by the comparative quantification of multiple calmodulin mRNAs in the rat brain by in situ hybridization with [35S]-cRNA probes. The procedure for quantification provides a significant improvement in that the direct and exact comparison of radiolabeled species, even from different experiments, can be reliably performed. Further, the procedure can be adapted to the quantification of autoradiographs produced by other methods.

Original languageEnglish
Pages (from-to)1141-1149
Number of pages9
JournalJournal of Histochemistry and Cytochemistry
Volume46
Issue number10
DOIs
Publication statusPublished - Oct 1998

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Keywords

  • Calibration procedure
  • Calmodulin mRNAs
  • Computer-assisted microdensitometry
  • Exponential function
  • Hyperfilm- βmax
  • Image analysis
  • In situ hybridization
  • Mathematical model
  • Quantitative autoradiography
  • S standards

ASJC Scopus subject areas

  • Anatomy
  • Histology

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