Transcription profiling by array of human LHK2 lung adenocarcinoma side population and main population cells

We used the commercially available amino-allyl RNA amplification Kit ver,2 (High Yield Type) (SIGMA-ALDRICH). Purified total RNA (3 µg) was reverse-transcribed to generate double-stranded cDNA using an oligo dT T7 promoter primer and reverse transcriptase. Next, cRNA was synthesized using T7 RNA polymerase, which simultaneously incorporated Cy3- or Cy5-labeled cytidine triphosphate. During this process, the samples of SP cells were labeled with Cy5, whereas the non-SP cells were labeled with Cy3 as control cells. Quality of the cRNA was again checked using the Nano Drop. Cy3-labeled cRNA and Cy5-labeled cRNA were combined and then fragmented in a hybridization cocktail (SIGMA-ALDRICH). Then the labeled cRNAs were hybridized to a 60-mer probe oligonucleotide microarray and incubated for 20 h ours at 50°C. The fluorescent intensities were determined by a Genepix 4000B Microarray Scanner (Axon, US).

本研究采用商用氨基烯丙基RNA扩增试剂盒（High Yield Type）第2版（SIGMA-ALDRICH）。取3 μg纯化后的总RNA，以寡聚dT T7启动子引物结合反转录酶进行反转录，合成双链互补DNA（cDNA）。随后利用T7 RNA聚合酶合成互补RNA（cRNA），反应过程中同时掺入Cy3或Cy5标记的三磷酸胞苷。此步骤中，侧群细胞（SP cells）样本采用Cy5进行荧光标记，而非侧群细胞作为对照样本则以Cy3标记。再次使用Nano Drop对cRNA的质量进行检测验证。将Cy3标记与Cy5标记的cRNA混合后，置于SIGMA-ALDRICH品牌的杂交缓冲液中进行片段化处理。随后将标记完成的cRNA与60聚体寡核苷酸探针微阵列进行杂交，于50℃条件下孵育20小时。荧光信号强度通过Genepix 4000B型微阵列扫描仪（Axon，美国）进行定量检测。