cell line: A549 cell type: lung carcinoma cell line transduced with pBabe vector control genotype/variation: LKB1-null
Treatment protocol
(A) Two groups: LKB1-null, LKB1-wt; (B) Three groups: Control (vector), LKB1 (wt), LKB1 kinase-death (K78I).
Growth protocol
Cells were cultured in Dulbecco's modified Eagle's medium (Mediatech, Manassa, VA) supplemented with 10% inactivated fetal bovine serum (Life Technologies, Carlsbad, CA) and 1% penicillin/streptomycin (Corning Cellgro, Manassa, VA). Cells were grown in a 5% CO2 cell culture incubator at 37 °C. For retroviral production, the retroviral pBabe-based construct (LKB1, kinase death mutant LKB1 K78I or empty vector) together with packing plasmid pMD.MLV and pseudotyped envelope pMD2-VSV-G were transfected to 293T cells using Effectene transfection reagent (QIAGEN, Germantown, MD). Retroviruses were collected at 48 hours and 72 hours post transfection and used to infect the A549 cells on two consecutive dates. Cells were collected for RNA and proteins at 96 hours post infection.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using TRIzol (Life Technologies, Carlsbad, CA) and RNeasy mini kit (Qiagen, Germantown, MD).
Label
Cy3
Label protocol
Sample labeling was performed based on the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar).
Hybridization protocol
The labeled cRNAs were hybridized onto the Agilent-045997 Human LncRNA Array v3.0 (8 x 60K, Arraystar).
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner G2505C using one color scan setting.
Description
LW5 LncRNA expression data from LKB1-null lung cancer A549 cells transduced with pBabe vector control.
Data processing
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 4 out of 10 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis.
