Ares Manuel
Cold Spring Harb Protoc. 2014 Jan 1;2014(1):119-23. doi: 10.1101/pdb.prot080127.
This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.
本方案描述了如何制备用于与微阵列杂交的荧光标记cDNA。它包括两个步骤：首先，使用锚定的寡聚（dT）和随机六聚体混合物作为引物，以带有反应性胺基的修饰脱氧核苷酸（氨基烯丙基-dUTP）和RNA样品为模板，通过逆转录酶进行胺修饰的cDNA合成。其次，纯化cDNA并将其置换到碳酸氢盐缓冲液中，使cDNA中的胺基与染料N-羟基琥珀酰亚胺（NHS）酯反应，将染料共价连接到cDNA上。再次纯化染料偶联的cDNA，并确定每微克cDNA掺入的染料量。
