Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer.
Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer.
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DOI：
10.1186/1471-2199-8-25
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发表时间：
2007-03-21
影响因子：
--
通讯作者：
Klee GG
中科院分区：
文献类型：
--
作者：
Kube DM;Savci-Heijink CD;Lamblin AF;Kosari F;Vasmatzis G;Cheville JC;Connelly DP;Klee GG
为了发现前列腺癌的生物标志物，我们分析了前列腺组织和转移性前列腺腺癌的良性和恶性细胞激光捕获微解剖（LCM）的基因表达。在这里，我们提出了开发、优化和验证的方法，以获得高质量的基因表达数据。将RNase抑制剂加入到用于LCM冷冻组织切片染色的溶液中，可以显著提高RNA质量。定量PCR检测，需要最少量的LCM RNA，以确定RNA的质量和浓度。SuperScript II™逆转录酶替换为SuperScript III™，消除SpeedVac浓度以优化线性扩增。使用GeneChip®IVT标记试剂盒而不是Enzo BioArray™HighYield™RNA转录物标记试剂盒，因为并排比较表明后者具有高端信号饱和度。总RNA线性扩增约2 ng后，平均得到72 μg的标记互补RNA。无监督聚类将5/5的正常前列腺增生和2/2的良性前列腺增生放在一组，5/7的Gleason 3型病例放在另一组，剩下的2/7的3型病例放在第三组，8/8的Gleason 5型病例和3/3的转移性前列腺腺癌。定量PCR证实α -甲基酰基辅酶A消旋酶（AMACR）和hepsin的差异表达。
To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data. RNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II™ reverse transcriptase was replaced with SuperScript III™, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip® IVT labeling kit was used rather than the Enzo BioArray™ HighYield™ RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 μg of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA. Unsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR) and hepsin was confirmed using quantitative PCR.
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影响因子：
14.9
作者：
Baugh, L. R.;Hill, A. A.;Hunter, Craig P.
通讯作者：
Hunter, Craig P.
影响因子：
14.9
作者：
Petalidis, L;Bhattacharyya, S;Lyons, PA
通讯作者：
Lyons, PA
影响因子：
6
作者：
Ernst, T;Hergenhahn, M;Gröne, HJ
通讯作者：
Gröne, HJ
影响因子：
2.7
作者：
Ohyama, H;Zhang, X;Todd, R
通讯作者：
Todd, R
DOI：
10.1073/pnas.89.7.3010
发表时间：
1992-04-01
影响因子：
11.1
作者：
EBERWINE, J;YEH, H;COLEMAN, P
通讯作者：
COLEMAN, P