Statistical Considerations in the Assessment of cDNA Microarray Data Obtained Using Amplification
Statistical Considerations in the Assessment of cDNA Microarray Data Obtained Using Amplification
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DOI：
10.1007/0-387-26288-1_2
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发表时间：
2006
期刊：
影响因子：
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通讯作者：
Jing Wang;K. Coombes;K. Baggerly;Limei Hu;S. Hamilton;Wei Zhang
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文献类型：
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作者：
Jing Wang;K. Coombes;K. Baggerly;Limei Hu;S. Hamilton;Wei Zhang
数据采集是微阵列研究基因表达谱的关键第一步。成功的数据采集取决于获得高于微阵列上背景噪声水平的一致信号。许多因素影响从微阵列实验中获得的荧光信号的质量和数量，包括荧光染料的强度、检测信号的扫描仪的灵敏度以及应用于微阵列的标记cDNA靶的量。为了增加信号强度，已经进行了大量的努力来开发更强的染料并提高扫描仪的灵敏度。已经开发了基于激光和CCD的扫描仪，但是大多数商业上可获得的扫描仪具有类似的性能（Ramdas等人，2001年）。染料和扫描仪本身的技术改进为使用少量起始材料从微阵列实验中产生强的、可重复的信号的问题提供了不完整的解决方案。微阵列数据采集中最重要的一个因素是所需RNA的量。为了获得一致的杂交和足够的信号，常规方案需要超过50μg的总RNA（或1-2 μg的mRNA）作为起始材料，通过在逆转录过程中掺入Cy 3-dNTP或Cy 5-dNTP来从其产生标记的cDNA靶标（图2.1）。为了获得如此数量的RNA，
Data acquisition is the critical first step in microarray studies to profile gene expression. Successful data acquisition depends on obtaining consistent signals above the level of the background noise on the microarray. Many factors influence the quality and quantity of the fluorescent signals obtained from microarray experiments, including the strength of the fluorescent dyes, the sensitivity of the scanners that detect the signals, and the amount of labeled cDNA targets applied to the microarray. In order to increase signal intensity, substantial efforts have been made to develop stronger dyes and to improve the sensitivity of the scanners. Both laser and CCD based scanners have been developed, but most commercially available scanners have similar performances (Ramdas et al., 2001). Technological improvements in dyes and scanners, by themselves, have provided incomplete solutions to the problem of generating strong, reproducible signals from microarray experiments using small amounts of starting materials.The single most important factor in microarray data acquisition is the amount of RNA required. In order to obtain consistent hybridizations and adequate signals, conventional protocols require more than 50μg of total RNA (or 1–2 μg of mRNA) as starting material, from which labeled cDNA targets are generated by incorporating Cy3-dNTP or Cy5-dNTP during reverse transcription (Fig. 2.1). To harvest this amount of RNA, several