Transcription profiling by array of human LHK2 lung adenocarcinoma side population and main population cells
ABSTRACT: 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).
ORGANISM(S): Homo sapiens
PROVIDER: S-ECPF-MEXP-3913 | BioStudies |