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Additional info for Molecular Methods in Developmental Biology: Xenopus & Zebrafish (Methods in Molecular Biology Vol 127)
Biol. 37, 291–298. 9. , and Takahashi, Y. (1997) Mesodermal subdivision along the mediolateral axis in the chicken controlled by different concentrations of BMP-4. Development 124, 1975–1984. 10. , and Takeda, H. (1997) Specification of posterior midbrain region in zebrafish neuroepithelium. Genes Cells 1, 369–377. 11. Tung, T. , Chang, C. , and Tung, Y. F. Y. (1945) Experiments on the developmental potencies of blastoderms and fragments of Teleostean eggs separated latitudianally. Proc. Zool. Sci.
Introduction When characterizing the developmental expression of a novel gene, or when examining the response of a known gene to experimental manipulations, it is important to be able to assay mRNA transcript levels accurately. Although a number of techniques for transcript analysis are available, one of the most useful and widespread is the ribonuclease (RNase) protection assay (for example, see Fig. 1). The major advantages of RNase protection analysis are good sensitivity, excellent specificity, and the linear response to differing transcript levels.
9. Incubate at 37°C for 30 min. 10. RNase inactivation is accomplished by the addition of 10 µL of 10% SDS and 2 µL of Proteinase K solution, followed by incubation for 15 min at 37°C. 11. Extract all samples with an equal volume of phenol–chloroform. 12. 5 Ribonuclease Protection Analysis 35 volumes of ethanol and 10 µg of carrier RNA. Salt is already present in the digestion buffer. 13. Recover the RNA by centrifugation for 10 min in a microfuge and then resuspend the pellet in 5 µL of formamide loading buffer.