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A method for rapid demineralization of teeth and bones.

Cho A, Suzuki S, Hatakeyama J, Haruyama N, Kulkarni AB - Open Dent J (2010)

Bottom Line: The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate.This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest.However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase.

View Article: PubMed Central - PubMed

Affiliation: Gene Targeting Facility, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA.

ABSTRACT
Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

No MeSH data available.


Related in: MedlinePlus

Demineralization at higher temperature does not affect nucleic acid stability.RT-PCR assay of Gapdh mRNA extracted from the slides of frozen tooth sections made from skulls decalcified at different temperatures; RT, 37°C, 42°C, and 50°C. The amplification of RNA confirms the stability of nucleic acids exposed to 50°C temperature during the demineralization process.
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Figure 7: Demineralization at higher temperature does not affect nucleic acid stability.RT-PCR assay of Gapdh mRNA extracted from the slides of frozen tooth sections made from skulls decalcified at different temperatures; RT, 37°C, 42°C, and 50°C. The amplification of RNA confirms the stability of nucleic acids exposed to 50°C temperature during the demineralization process.

Mentions: Elevated incubation temperature used for demineralization process did not affect stability of nucleic acids: To evaluate the effects of elevated temperatures on the stability of nucleic acids, we performed RT-PCR using mRNA extracted from the tooth sections. The amplification of Gapdh mRNA transcripts at 37°C, 42˚C or 50°C all showed similar levels (Fig. 7).


A method for rapid demineralization of teeth and bones.

Cho A, Suzuki S, Hatakeyama J, Haruyama N, Kulkarni AB - Open Dent J (2010)

Demineralization at higher temperature does not affect nucleic acid stability.RT-PCR assay of Gapdh mRNA extracted from the slides of frozen tooth sections made from skulls decalcified at different temperatures; RT, 37°C, 42°C, and 50°C. The amplification of RNA confirms the stability of nucleic acids exposed to 50°C temperature during the demineralization process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040998&req=5

Figure 7: Demineralization at higher temperature does not affect nucleic acid stability.RT-PCR assay of Gapdh mRNA extracted from the slides of frozen tooth sections made from skulls decalcified at different temperatures; RT, 37°C, 42°C, and 50°C. The amplification of RNA confirms the stability of nucleic acids exposed to 50°C temperature during the demineralization process.
Mentions: Elevated incubation temperature used for demineralization process did not affect stability of nucleic acids: To evaluate the effects of elevated temperatures on the stability of nucleic acids, we performed RT-PCR using mRNA extracted from the tooth sections. The amplification of Gapdh mRNA transcripts at 37°C, 42˚C or 50°C all showed similar levels (Fig. 7).

Bottom Line: The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate.This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest.However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase.

View Article: PubMed Central - PubMed

Affiliation: Gene Targeting Facility, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA.

ABSTRACT
Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

No MeSH data available.


Related in: MedlinePlus