Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study.
Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study.
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DOI：
10.1186/1471-2164-5-26
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发表时间：
2004-04-27
期刊：
影响因子：
4.4
通讯作者：
Zilberstein A
中科院分区：
文献类型：
--
作者：
Cao W;Epstein C;Liu H;DeLoughery C;Ge N;Lin J;Diao R;Cao H;Long F;Zhang X;Chen Y;Wright PS;Busch S;Wenck M;Wong K;Saltzman AG;Tang Z;Liu L;Zilberstein A
几种高通量技术已被用于鉴定可能成为药物发现分子靶点的差异调控基因。在这里，我们比较了使用两种实验方法发现的差异调节基因集：减法抑制杂交（SSH） cDNA文库方法和Affymetrix GeneChip®技术。在这个“案例研究”中，我们探索了人类单核细胞向髓系树突状细胞（DC）体外分化过程中的转录模式变化，并评估了使用SSH方法发现新基因的潜力。从外周血单核细胞前体和未成熟DC （iDC）中分离出相同的RNA样本，用于基因芯片探测和SSH cDNA文库构建。从每个双向SSH库（iDC-monocytes和单核细胞- idc）中挑选10,000个克隆进行测序。每个文库从8000个成功序列中鉴定出约2000个转录本。只有70%到75%的转录本在U95系列基因芯片上显示，这意味着25%到30%的转录本可能无法在仅基于基因芯片的研究中被识别。此外，这些转录本中约有10%似乎是“新颖的”，尽管这些转录本尚未经过仔细研究。在芯片上显示的转录本中，通过GeneChip微阵列转录本分析，一致发现约三分之一的转录本在iDC和单核细胞之间存在差异调控。剩下的三分之二要么没有从GeneChip微阵列数据推断为差异调节，要么被称为差异调节，但方向相反。这强调了生成对等的SSH库对以及对结果进行实时RT-PCR确认的重要性。该研究表明，SSH可以作为基因芯片的替代和补充转录物分析工具，特别是在鉴定新基因和低丰度转录物方面。
Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip® technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology. The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results. This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.
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影响因子：
3.4
作者：
Evans, SJ;Datson, NA;Akil, H
通讯作者：
Akil, H
影响因子：
15.3
作者：
Romani, Nikolaus;Gruner, Stefan;Brang, Daniela;Kaempgen, Eckhart;Lenz, Angela;Trockenbacher, Bettina;Konwalinka, Guenther;Fritsch, Peter O.;Steinman, Ralph M.;Schuler, Gerold
通讯作者：
Schuler, Gerold
影响因子：
56.9
作者：
LISITSYN, N;LISITSYN, N;WIGLER, M
通讯作者：
WIGLER, M
影响因子：
8
作者：
Wang, T;Hopkins, D;Reed, SG
通讯作者：
Reed, SG
DOI：
10.1073/pnas.93.12.6025
发表时间：
1996-06-11
影响因子：
11.1
作者：
Diatchenko, L;Lau, YFC;Siebert, PD
通讯作者：
Siebert, PD