Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.
Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.
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制备激光捕获显微解剖的组织学染色方法显着影响细胞RNA的完整性。
DOI：
10.1186/1471-2164-7-97
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发表时间：
2006-04-27
期刊：
影响因子：
4.4
通讯作者：
Mushinski JF
中科院分区：
文献类型：
--
作者：
Wang H;Owens JD;Shih JH;Li MC;Bonner RF;Mushinski JF
通过激光捕获显微切割（LCM）富集的细胞的微阵列分析的基因表达谱面临几个技术挑战。冷冻切片比石蜡包埋切片产生更高质量的RNA，但即使使用冷冻切片，用于组织学鉴定感兴趣细胞的染色方法仍然可能损害细胞中的mRNA。为了研究染色方法对LCM样品基因表达谱结果降解的贡献，我们将小鼠浆细胞肿瘤细胞系TEPC 1165的颗粒进行直接RNA提取，并进行平行冷冻切片以进行LCM和随后的RNA提取。我们使用微阵列杂交分析来比较来自细胞团的RNA的基因表达谱与来自冷冻切片的RNA的基因表达谱，所述冷冻切片已经用苏木精和伊红（H&E）、尼氏染色（NS）和免疫荧光（IF）以及用浆细胞揭示甲基绿色派洛宁（MGP）染色染色。所有的RNA扩增与两轮的T7为基础的体外转录和双色表达分析10-K cDNA微阵列上进行分析。MGP染色的样品显示出最少的mRNA损失，其次是H&E和免疫荧光。尼氏染色是显着更有害的基因表达谱，大概是由于水的步骤中，RNA可能已被内源性或外源性RNA酶损伤。RNA损伤可能发生在准备激光捕获显微切割的染色步骤期间，其结果是在微阵列杂交分析中某些基因的代表性丧失。在水性染色溶液中包含RNA酶抑制剂似乎在保护RNA免受基因转录物损失方面是重要的。
Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.
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影响因子：
2.7
作者：
Feldman, AL;Costouros, NG;Libutti, SK
通讯作者：
Libutti, SK
影响因子：
11.6
作者：
Nakazono, M;Qiu, F;Schnable, PS
通讯作者：
Schnable, PS
影响因子：
32.4
作者：
Shaffer, AL;Lin, KI;Staudt, LM
通讯作者：
Staudt, LM
影响因子：
56.9
作者：
NORDAN, RP;POTTER, M
通讯作者：
POTTER, M
影响因子：
6
作者：
Fend, F;Emmert-Buck, MR;Raffeld, M
通讯作者：
Raffeld, M