Advanced Biomedical Research
Extraction of total RNA in the developing chicken forebrain
Authors
Department of Biology, University of Isfahan, Isfahan, Iran
Abstract
Background: Gene expression of Gama-Aminobutyric acid (GABA A ) receptor subunits may change during development. Procedures in molecular biology are required to understand the gene expression profile GABA A R in chicken. The outcome of the results depends on good-quality high-molecular-weight RNA. Several procedures can be used to isolate RNA from the brain of chicken; however, most of them are time-consuming and require disruption of cells or freeze and thaw in the presence of RNase inhibitors. The aim of this experiment was isolation of RNA from chicken embryonic brain tissues using appropriate RNA extraction kit.
Materials and Methods: Fertilized eggs from Ross breed (Gallus gallus) were incubated at 38°C and 60% relative humidity in a forced-draft incubator and were turned every 3 h. After 3, 7, 14 and 20 days of incubation, eggs were cooled on ice to induce deep anesthesia. Then whole brains were dissected out. As brains could not be excised in a reproducible way from earlier embryos (embryonic days 4 and 6), whole heads were collected. Chicken embryos between day 7 to 20 and 1 day after birth were decapitated, and their brains removed. Samples were immediately inserted into lysis buffer and stored at −70°C. Total RNA was isolated and a contaminating genomic deoxyribonucleic acid (DNA) was digested. RNA quality was checked using gel electrophoresis.
Results: We obtained 52 mg/ml to 745 mg/ml with A260/280 1.7-2.2. Only high-quality RNA, with no signs of degradation, was used for further experiments.
Conclusion: In conclusion, protocol was found to be suitable for the isolation of total RNA from embryonic chicken cells.
Keywords
| 1. | Barnard EA, Skolnick P, Olsen RW, Mohler H, Sieghart W, Biggio G, et al. International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acid A receptors: Classification on the basis of subunit structure and receptor function. Pharmacol Rev 1998;50:291-313.  |
| 2. | Olsen RW, Tobin AJ. Molecular biology of GABAA receptors. FASEB J 1990;4:1469-80. [PUBMED] |
| 3. | Sieghart W, Sperk G. Subunit composition, distribution and function of GABA (A) receptor subtypes. Curr Top Med Chem 2002;2:795-816. [PUBMED] |
| 4. | Mohler H. GABA (A) receptor diversity and pharmacology. Cell Tissue Res 2006;326:505-16. |
| 5. | Rudolph U, Möhler H. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr Opin Pharmacol 2006;6:18-23. |
| 6. | Enomoto K, Kataoka H, Hirota A. Semi quantitative analysis of the expression of GABA-A receptor subunits in the developing embryonic chick brain stem. Jpn J Physiol 2001;51:53-61. [PUBMED] |
| 7. | Leboy PS, Shapiro IM, Uschmann BD, Oshima O, Lin D. Gene expression in mineralizing chick epiphyseal cartilage. J BiolChem 1988;263: 8515-20. |
| 8. | Jackwood MW, Kwon HM, Hilt DA. Infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a DNA probe. Avian Dis 1992;36:403-9. [PUBMED] |
| 9. | Zaker SR, Esmaeili A, Bouzari M, Shirani E. Quantitative Analysis of GABAA Gamma Receptor Subunits in the Developing Embryonic Chick Forebrain.Iran J Basic Med Sci 2012;15:1097-101. [PUBMED] |
| 10. | Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 2008;454:49-55. [PUBMED] |
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