Abstract
It is extremely useful in investigations of the central nervous system (CNS) to measure mRNA expression in cells by in situ hybridization. However, this approach is limited by the difficulties of a reliable quantitative evaluation. In the present paper we describe a method for quantifying radioactive hybrids on individual cells by a silver grain count in digital images. Quantification is based on the real size and grey level of a single grain obtained by computerized microscope image analysis (IBAS 2, Kontron-Zeiss, PC 286). The program provides for automatic identification of cell area, the portion occupied by grains and their grey level. The number of grains per cell results from a mathematical function integrating these parameters with a 'density factor'. This factor is introduced to better estimate the number of grains when there is overlapping. The method was tested by measuring the expression of preproNPY (pp-NPY) mRNA in rat dentate gyrus and preproSomatostatin (pp-SOM) mRNA in frontal cerebral cortex of control and colchicine-treated rats. Colchicine did not modify the number of pp-SOM mRNA-positive cells but reduced the expression per cell. These results confirm the advantages of our method to quantify a wide range of silver grains (10-5000) and improves the sensitivity of in situ hybridization. With this support the in situ hybridization technique could be considered a real quantitative method for measuring small alterations in neuronal function.
| Original language | English |
|---|---|
| Pages (from-to) | 93-103 |
| Number of pages | 11 |
| Journal | Journal of Neuroscience Methods |
| Volume | 47 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - 1993 |
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Keywords
- Autoradiography
- Central nervous system
- Computerized microscope image analysis
- mRNA expression
- PreproNPY mRNA
- Somatostatin
ASJC Scopus subject areas
- Neuroscience(all)
Cite this
In situ hybridization histochemistry quantification : automatic count on single cell in digital image. / Masseroli, M.; Bollea, A.; Bendotti, C.; Forloni, G.
In: Journal of Neuroscience Methods, Vol. 47, No. 1-2, 1993, p. 93-103.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - In situ hybridization histochemistry quantification
T2 - automatic count on single cell in digital image
AU - Masseroli, M.
AU - Bollea, A.
AU - Bendotti, C.
AU - Forloni, G.
PY - 1993
Y1 - 1993
N2 - It is extremely useful in investigations of the central nervous system (CNS) to measure mRNA expression in cells by in situ hybridization. However, this approach is limited by the difficulties of a reliable quantitative evaluation. In the present paper we describe a method for quantifying radioactive hybrids on individual cells by a silver grain count in digital images. Quantification is based on the real size and grey level of a single grain obtained by computerized microscope image analysis (IBAS 2, Kontron-Zeiss, PC 286). The program provides for automatic identification of cell area, the portion occupied by grains and their grey level. The number of grains per cell results from a mathematical function integrating these parameters with a 'density factor'. This factor is introduced to better estimate the number of grains when there is overlapping. The method was tested by measuring the expression of preproNPY (pp-NPY) mRNA in rat dentate gyrus and preproSomatostatin (pp-SOM) mRNA in frontal cerebral cortex of control and colchicine-treated rats. Colchicine did not modify the number of pp-SOM mRNA-positive cells but reduced the expression per cell. These results confirm the advantages of our method to quantify a wide range of silver grains (10-5000) and improves the sensitivity of in situ hybridization. With this support the in situ hybridization technique could be considered a real quantitative method for measuring small alterations in neuronal function.
AB - It is extremely useful in investigations of the central nervous system (CNS) to measure mRNA expression in cells by in situ hybridization. However, this approach is limited by the difficulties of a reliable quantitative evaluation. In the present paper we describe a method for quantifying radioactive hybrids on individual cells by a silver grain count in digital images. Quantification is based on the real size and grey level of a single grain obtained by computerized microscope image analysis (IBAS 2, Kontron-Zeiss, PC 286). The program provides for automatic identification of cell area, the portion occupied by grains and their grey level. The number of grains per cell results from a mathematical function integrating these parameters with a 'density factor'. This factor is introduced to better estimate the number of grains when there is overlapping. The method was tested by measuring the expression of preproNPY (pp-NPY) mRNA in rat dentate gyrus and preproSomatostatin (pp-SOM) mRNA in frontal cerebral cortex of control and colchicine-treated rats. Colchicine did not modify the number of pp-SOM mRNA-positive cells but reduced the expression per cell. These results confirm the advantages of our method to quantify a wide range of silver grains (10-5000) and improves the sensitivity of in situ hybridization. With this support the in situ hybridization technique could be considered a real quantitative method for measuring small alterations in neuronal function.
KW - Autoradiography
KW - Central nervous system
KW - Computerized microscope image analysis
KW - mRNA expression
KW - PreproNPY mRNA
KW - Somatostatin
UR - http://www.scopus.com/inward/record.url?scp=0027215791&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027215791&partnerID=8YFLogxK
U2 - 10.1016/0165-0270(93)90025-M
DO - 10.1016/0165-0270(93)90025-M
M3 - Article
VL - 47
SP - 93
EP - 103
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 1-2
ER -