Methods for quantification of in situ hybridization signals obtained by film autoradiography and phosphorimaging applied for estimation of regional levels of calmodulin mRNA classes in the rat brain

Sandor Vizi, Arpad Palfi, Laszlo Hatvani, Karoly Gulya

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Comparative analysis of the regional abundances of the various mRNAs in neural tissues requires the quantitation of target nucleic acid sequences while their tissue distribution is preserved. A quantitative in situ hybridization protocol is presented for the assessment of regional levels of calmodulin (CaM) I, II and III mRNAs in the rat brain. Coronal brain cryostat sections were hybridized with multiple CaM [35S]cRNA probes and co-exposed to an autoradiographic film or storage phosphor screen, together with a membrane-based radioactive standard scale. The membrane scale was calibrated against a brain paste standard scale. Regression analyses of the sensitometric graphs of standard scales corresponding to the autoradiographic film and to the storage phosphor screen were performed by means of exponential (ROD=p1(1-exp[-p2x])) and linear (LI=ax) functions, respectively (ROD is relative optical density, LI is labeling intensity, and x is radioactivity). The ROD/LI values for the hybridized brain regions were converted into cRNA probe copy numbers (estimations of mRNA copy numbers) through use of the above standard scales. This method was applied to compare the regional abundances of multiple CaM mRNAs in the brains of control, dehydrated, chronic ethanol-treated and ethanol withdrawal-treated animals.

Original languageEnglish
Pages (from-to)32-44
Number of pages13
JournalBrain Research Protocols
Volume8
Issue number1
DOIs
Publication statusPublished - Jan 1 2001

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Keywords

  • Calmodulin mRNA
  • Film autoradiography
  • Gene expression
  • Image analysis
  • Phosphorimaging
  • Quantitative in situ hybridization
  • Rat brain

ASJC Scopus subject areas

  • Neuroscience(all)

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