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首页> 外文期刊>BMC Biotechnology >Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range
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Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range

机译：自己动手：构建具有高灵敏度和线性范围的定制cDNA宏阵列平台

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Background Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA -based nylon macroarrays as well as sample 33P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. Results The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p Conclusions Results presented here, demonstrate for the first time that self-made cDNA -based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner.
机译：涉及基因表达谱分析和临床应用（例如诊断和预后）的背景研究通常需要可灵活定制和具有成本效益的DNA阵列平台，但同时具有高度敏感性并能够准确，可复制地定量DNA的转录表达。使用少量RNA样品可在整个转录组动态范围内获得大量基因。到目前为止，对基于cDNA的尼龙宏阵列以及样品 33 P标记，杂交方案和荧光粉的设计进行了一系列易于实现的实际优化分析屏幕图像处理的宏阵列性能。结果本文提出的定制宏阵列平台的绝对灵敏度低至50,000个转录本，线性范围超过5个对数阶。鉴定差异表达基因的质量至少可与市售微芯片相媲美。有趣的是，发现定量准确度与相应的逆转录酶-定量PCR值，金标准基因表达量显着相关（皮尔逊相关性检验p结论）此处介绍的结果首次证明了基于cDNA的自制尼龙尼龙阵列可以产生具有高度敏感性的高度可靠的基因表达数据，并涵盖了哺乳动物mRNA丰度的整个动态范围，从每个样品的少量未扩增总RNA开始，可以同时分析合理数量的样品，以定量检测数百个基因。一种易于定制且具有成本效益的方式。

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