Calculation of maximal hybridization capacity (Hmax) for quantitative in situ hybridization: A case study for multiple calmodulin mRNAs

Sandor Vizi, K. Gulya

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

In estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CAM) I, II, and III [35S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [35S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm2 units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way.

Original languageEnglish
Pages (from-to)893-904
Number of pages12
JournalJournal of Histochemistry and Cytochemistry
Volume48
Issue number7
Publication statusPublished - Jul 2000

Fingerprint

Calmodulin
In Situ Hybridization
Messenger RNA
Brain
Calibration
Complementary RNA

Keywords

  • Calmodulin mRNAs
  • Gene expression
  • Hmax
  • Image analysis
  • Phosphorimager
  • Quantitative in situ hybridization
  • Rat brain

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

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abstract = "In estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CAM) I, II, and III [35S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [35S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm2 units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way.",
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AB - In estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CAM) I, II, and III [35S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [35S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm2 units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way.

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