Methacarn as a whole brains fixative for gene and protein expression analyses of specific brain regions in rats
Methacarn as a whole brains fixative for gene and protein expression analyses of specific brain regions in rats
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DOI：
10.2131/jts.38.431
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发表时间：
2013-06-01
影响因子：
2
通讯作者：
Shibutani, Makoto
中科院分区：
文献类型：
--
作者：
Akane, Hirotoshi;Saito, Fumiyo;Shibutani, Makoto
为了在啮齿类动物的解剖特异性脑区进行分子分析，有必要建立一种快速准确的组织采样程序，以实现提取分子的高完整性和表达保真度。本研究的目的是检验甲沙卡恩全脑固定的适用性，并随后使用穿孔活检装置进行组织取样，用于大鼠基因表达分析。固定后，采集海马齿状体、胼胝体、扣带皮层或小脑蚓部等特定区域，检测提取总rna和多肽表达数据的完整性和可变性。比较Methacarn固定、丙酮固定和未固定组织。Methacarn固定导致总rna的高完整性，足以进行全局表达分析，并且在脑区域完整性的均匀性方面优于丙酮固定。经甲沙卡恩固定提取的多肽与未固定或丙酮固定提取的多肽具有相似的完整性。在微阵列分析中，与丙酮固定相比，甲基活性炭固定导致样品间mRNA表达变异性较低。在二维差分凝胶电泳中，甲基沙卡恩和丙酮固定的多肽表达保真度基本相当，尽管丙酮固定的组织中少数多肽的表达水平受到影响。这些结果表明，在啮齿类动物研究中，全脑固定使用甲基沙坎对mrna和多肽的全局分析具有优势。
For molecular analysis in anatomically-specific brain regions for rodent studies, it is necessary to establish a fast and accurate procedure for tissue sampling to achieve high integrity and expression fidelity of extracted molecules. The present study was performed to examine suitability of whole brain fixation with methacarn and subsequent tissue sampling using punch-biopsy devices for gene expression analysis in rats. After fixation, each specific region, i.e., hippocampal dentate gyms, corpus callosum, cingulate cortex or cerebellar vermis was collected, and the integrity and variability of expression data of extracted total RNAs and polypeptides were examined. Methacarn fixation, acetone fixation, and unfixed tissues were compared. Methacarn fixation resulted in high integrity of total RNAs sufficient for conducting global expression analysis and superior in terms of uniformity in the integrity among brain regions to that of acetone fixation. Extracted polypeptide after methacarn fixation revealed similar integrity to that without fixation or with acetone fixation. Methacarn fixation resulted in lower mRNA expression variability between samples than acetone fixation in microarray analysis. The fidelity of polypeptide expression was mostly equivalent between methacarn and acetone fixation in 2-dimensional differential in-gel electrophoresis, although the expression levels of a small number of polypeptides from acetone-fixed tissues were affected. These results suggest that whole brain fixation with methacarn retains advantages for global analyses of mRNAs and polypeptides in rodent studies.