Fluorescent labelling of cRNA for microarray applications
Fluorescent labelling of cRNA for microarray applications
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DOI：
10.1093/nar/gng020
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发表时间：
2003-03-01
影响因子：
14.9
通讯作者：
den Dunnen, JT
中科院分区：
文献类型：
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作者：
't Hoen, PAC;de Kort, F;den Dunnen, JT
寡核苷酸表达文库的微阵列可以与在逆转录期间从mRNA产生的cDNA或在Eberwine mRNA扩增程序中产生的cRNA杂交。虽然cDNA的荧光标记方法已被彻底研究，但cRNA标记的方法还没有。为了这个目的，我们开发了一个aminoallyl-UTP（aa-UTP）驱动的cRNA标记协议，并比较了它在表达谱研究中使用点7.5 K 65聚体鼠寡核苷酸阵列通过直接掺入Cy-UTP标记。在AA-修饰的cRNA与N-羟基琥珀酰亚胺修饰的荧光Cy染料偶联期间，二甲亚砜的存在大大提高了标记效率，如通过分光光度法和荧光杂交信号分析的。使用aa-UTP的间接标记导致比通过直接掺入Cy-UTP的标记高2至3倍的标记和荧光信号。通过改变aa-UTP：UTP比率，发现了明确的最佳标记程度（1种染料/20-25 nt）。掺入更多的标记物增加了Cy 3信号，但降低了Cy 5荧光。这种效应可能是由于淬灭，这是更突出的Cy 5比Cy 3。总之，目前开发的方法是一种有效的，强大的和廉价的技术荧光标记的cRNA，并允许敏感的检测寡核苷酸微阵列上的基因表达谱。
Microarrays of oligonucleotide expression libraries can be hybridised with either cDNA, generated from mRNA during reverse transcription, or cRNA, generated in an Eberwine mRNA amplification procedure. While methods for fluorescent labelling of cDNA have been thoroughly investigated, methods for cRNA labelling have not. To this purpose, we developed an aminoallyl-UTP (aa-UTP) driven cRNA labelling protocol and compared it in expression profiling studies using spotted 7.5 K 65mer murine oligonucleotide arrays with labelling via direct incorporation of Cy-UTPs. The presence of dimethylsulfoxide during coupling of aa-modified cRNA with N-hydroxysuccinimide-modified, fluorescent Cy dyes greatly enhanced the labelling efficiency, as analysed by spectrophotometry and fluorescent hybridisation signals. Indirect labelling using aa-UTP resulted in 2- to 3-fold higher degrees of labelling and fluorescent signals than labelling by direct incorporation of Cy-UTP. By variation of the aa-UTP:UTP ratio, a clear optimal degree of labelling was found (1 dye per 20-25 nt). Incorporation of more label increased Cy3 signal but lowered Cy5 fluorescence. This effect is probably due to quenching, which is more prominent for Cy5 than for Cy3. In conclusion, the currently developed method is an efficient, robust and inexpensive technique for fluorescent labelling of cRNA and allows sensitive detection of gene expression profiles on oligonucleotide microarrays.