Abstract
We have developed a highly sensitive and reproducible method to detect the expression of specific genes in small tissue samples, such as a single embryonic somite. The procedure, which utilizes coupled reverse transcription-polymerase chain reaction (RT-PCR), was developed for evaluating the sequence of gene expression occurring in single somites during chick embryonic development. Comparisons of results obtained from using combinations of various RNA isolation methods and reverse transcription methods demonstrate that a protocol using a commercially available RNA isolation reagent (Tri Reagent) followed by optimized PCR, successfully detects low levels of mRNAs.
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References
Nusslein-Volhard, C., Frohnhofer, H. G., and Lehman, R. (1987) Determination of anterioposterior polarity in Drosophila.Science 238, 1675–1681.
Small, S. and Levine, M. (1991) The initiation of pairrule stripes in the Drosophila embryo.Curr. Opin. Genet. Devel. 1, 255–260.
Rosen, B. and Beddington, R. (1993) Whole mount in-situ hybridization in the mouse embryo: gene expression in three dimensions.Trends Genet. 9, 162–167.
Hsu, C. W. and Tuan, R. S. (1997) Whole mount in situ hybridization for developing chick embryos using digoxygenin-labeled RNA probes, inMethods in Molecular Biology, vol. 63, Recombinant Protein Protocols: Detection and Isolation (Tuan, R. S., ed.), Humana, Totowa, NJ, pp. 263–270.
Shepley, K. J. and Tuan, R. S. (1997) Combined, sequentialin situ hybridization and immunohistochemistry on the same tissue section, inMethods in Molecular Biology, vol. 63: Recombinant Protein Protocols: Detection and Isolation (Tuan, R. S., ed.), Humana, Totowa, NJ, pp. 247–256.
Krumlauf, R. (1994) Analysis of gene expression by northern blot.Mol. Biotechnol. 2, 227–242.
Lee, J. J. and Costlow, N. A. (1987) A molecular titration assay to measure transcript prevalence levels.Meth. Enzymol. 152, 643–648.
Gilman, M. (1989) Ribonuclease protection assay, inCurrent Protocols in Molecular Biology (Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K. eds.), Wiley, New York, pp. 4.7.1–4.9.16.
Masters, D. B., Griggs, C. T., and Berde, C. B. (1990) High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning.Biotechniques 12, 902–906.
Brown, T. and Mackey, K. (1989) Analysis of RNA by northern and slot blot hybridization, inCurrent Protocols in Molecular Biology (Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K., eds.), Wiley, New York, pp. 4.9.1–4.9.16.
Griffin, H. and Griffin, A. (1994)PCR Technology: Current Innovations. CRC, Boca Raton, FL.
Christ, B. and Ordahl, P. (1995) Early stages of chick somite development.Anat. Embryol. 191, 381–396.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning-A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Hamburger, V. and Hamilton, H. (1951) A series of normal stages in the development of the chick embryo.J. Morphol. 88, 49–92.
Chomcyzynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162, 156–159.
Dieffenbach, C. W., and Dveksler, G. S. eds. (1995)PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Barnes, G. L., Hsu, C. W., Mariani, B. D., and Tuan, R. S. (1996) Chicken Pax-1 gene: structure and expression during embryonic somite development.Differentiation 61, 13–23.
Peters, H., Doll, U., and Niessing, J. (1995) Differential expression of the chicken Pax-1 and Pax-9 genes; in-situ hybridization and immunohistochemical analysis.Develop. Dynam. 203, 1–10.
Kost, T. A., Theodorakis, N., and Hughes, S. H. (1983) The nucleotide sequence of the chick cytoplasmic beta-actin gene.Nucleic Acids Res. 11, 8287–8301.
Dugaiczyk, A., Haron, J. A., Stone, E. M., Dennison, O. E., Rothblum, K. N., and Schwartz, R. J. (1983) Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle.Biochemistry 22, 1605–1613.
Lin, Z. Y., Dechesne, C. A., Eldridge, J., and Patterson, B. M. (1989) An avian muscle factor related to MyoD1 activates muscle specific promotors in nonmuscle cells of different germ-layer origin and in BrdU treated myoblasts.Genes Devel. 3, 986–996.
Barnes, G. L., Alexander, P. G., Hsu, C. W., Mariani, B. D., and Tuan, R. S. (1997) Cloning and characterization of chicken paraxis: a regulator of paraxial mesoderm development and somite formation.Devel. Biol. 189, 95–111.
Nah, H. D. and Upholt, W. B. (1991) Type II collagen mRNA containing an alternatively spliced exon predominates in the chick limb prior to chondrogenesis.J. Biol. Chem. 266, 23,436–23,438.
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Sanzo, J.F., Tuan, R.S. High sensitivity analysis of gene expression in single embryonic somites using coupled reverse transcription-polymerase chain reaction. Mol Biotechnol 9, 7–15 (1998). https://doi.org/10.1007/BF02752693
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DOI: https://doi.org/10.1007/BF02752693