Transcription profiling of human miR-181a transfected K562 cells and mock transfected K562 cells

ABSTRACT: This series consists of samples taken from two groups of K562 cells(miR-181a transfected group and control group ) and harvested 48 hours later. We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Further studies aimed to find target mRNA of miR-181a in mammalian cells and its biological function. Experiment Overall Design: The K562 cells in our research were divided into tranfected group and mock-transfected control according to Lim et al. Experiment Overall Design: To transfect, we diluted 60 pmol miR-181 a duplex into 12 µL Opti-Mem each well, then we diluted 3 µL Oligofectamine(Invitrogen) into 48 µL Opti-Mem for each sample, incubated for 5 min at room temperature and add 10 µL of diluted transfection mixture to wells and incubate for another 20 min at room temperature. We next added 100 µL of diluted cells suspension mixture containing 50000 cells on top of the complex. Four hours after transfection, FBS was added to the cells at a final concentration of 10% and the cells were incubated for 48 hours. And control group was transfected only with Oligofectamine but no RNA. Each individual transfection experiment was performed in 10 replicate wells. Experiment Overall Design: After transfection experiments, total RNAs from above two group K562 cells were purified using the RNeasy mini kit (Qiagen, Heidelberg, Germany) in accordance with the manufacturer’s protocols. RNA concentration and purity were estimated spectrophotometrically and the quality further assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Experiment Overall Design: Total RNA from control group and transfected group were respectively reverse transcription, in vitro transcribed and labeled (Cy3 and Cy5) according to the Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol, green is control group, red is transfected group. The array hybridization, washing, and scanning procedures were performed in our lab according to Agilent 60-mer oligo microarray processing protocol using 0.75 µg samples of labeled, fragmented cRNA on Agilent Human 1A oligo microarray with 22575 probes (Agilent Technologies). Labeled cRNA is incubated with the microarray in a hybridization chamber at 60ºC for 17 hours, 4 rpm/min. The microarray was scanned using Agilent 2565BA DNA microarray scanner (Agilent Technologies). Data collection was performed by Agilent DNA microarray scanner. Using the Agilent Feature Extraction Software(v.7.5), spots were identified, measured for fluorescence intensity, local background, and arraywide parameters used to normalize the signals. Outliers and saturated spots were rejected according to 1.42xIQR, and local backgroud subtracted from the signals. Signals were then normalized to remove dye bias and arraywide drift in flourescence. Experiment Overall Design: The following two tests are performed to determined the significance of a feature: (1) Log ratio P-value of the red over green processed signals was calculated using two-sided t-test with P<0.01 considered significant, and (2) a boolean flag of 1 indicated that the feature BGSubSignal is well above background and passes the IsPosAndSignif test. Experiment Overall Design: We used Agilent human 1A oligo microarray identified the changes in gene expression profile of K562 cells after miR-181a transfection experiment. Experiment Overall Design: Further studies aimed to find target mRNA of miR-181a in mammalian cells and the function of miR-181a in cancers.

ORGANISM(S): Homo sapiens

SUBMITTER: Jueyu Zhou 

PROVIDER: E-GEOD-3605 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress