Comment[ArrayExpressAccession] E-GEOD-65263 MAGE-TAB Version 1.1 Public Release Date 2015-12-31 Investigation Title LncRNA in pediatric AML vs normal bone marrow mononuclear cells Comment[Submitted Name] LncRNA in pediatric AML vs normal bone marrow mononuclear cells Experiment Description Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common form of leukemia in children. Multiple lncRNAs participate in normal and may be implicated in malignant hematopoiesis associated with blood cell cancers, such as leukemia. Currently, the expression profile of lncRNAs in pediatric AML is unclear. In this study, we evaluated the lncRNA expression profile in the tissue of three pediatric AML patients with lncRNA microarray techniques. In order to gain insight into the function of targets of lncRNAs, GO term and KEGG pathway annotation were applied to the target gene pool. qPCR was performed to evaluate the expression patterns of dys-regulated lncRNAs. Bone marrow specimens were obtained at the time of diagnosis during routine clinical assessment of 3 pediatric patients with AML, who presented at the Department of Hematology and Oncology, Children's Hospital of Soochow University between 2000 and 2011. Additionally, bone marrow samples from 3 healthy donors were analyzed as controls. Human LncRNA Array analysis was performed by KangChen Bio-tech, Shanghai P.R. China. Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. For microarray analysis, Agilent Array platform was employed. The sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols 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. The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (8 x 60K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. 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 v12.0 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 4 out of 6 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Pathway analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show the distinguishable LncRNAs and mRNAs expression pattern among samples. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Pan Cao Pan Person First Name Jian Lan Jian Person Email panjian2008@163.com Person Affiliation Children's Hospital of Soochow University Person Phone 86-512-67786601 Person Address Department of Hematology and Oncology, Children's Hospital of Soochow University, Jingde road 303, suzhou, China Person Roles submitter Protocol Name P-GSE65263-1 P-GSE65263-4 P-GSE65263-5 P-GSE65263-2 P-GSE65263-3 P-GSE65263-6 Protocol Description Agilent Feature Extraction software (version 10.7.3.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). After quantile normalization of the raw data, lncRNAs and mRNAs that at least 3 out of 15 samples had flagged as “Present” or “Marginal” were chosen for further data analysis. Differentially expressed lncRNAs and mRNAs were identified through fold change (FC) filtering. Differentially expressed lncRNAs and mRNAs with statistical significance (as determined by two-tailed student’s t-test < 0.05) were identified through Volcano Plot filtering. Hierarchical Clustering was performed using the Agilent GeneSpring GX software (version 11.5.1). ID_REF = VALUE = normalized signal intensity Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) Bone marrow specimens were obtained at the time of diagnosis during routine clinical assessment of 3 pediatric patients with AML, who presented at the Department of Hematology and Oncology, Children's Hospital of Soochow University between 2000 and 2011. Additionally, bone marrow samples from 3 healthy donors were analyzed as controls.Bone marrow mononuclear cells (BMNCs) were isolated using Ficoll solution within 2 h after bone marrow samples harvested and subjected for the extraction of total RNA. Human LncRNA Array analysis was performed by KangChen Bio-tech, Shanghai P.R. China. Briefly, RNA purified from total RNA after removal of rRNA was amplified and transcribed into fluorescent cRNA and cDNA was labeled and hybridized to the Human LncRNA Array v3.0 (8660 K, Arraystar). 30,586 LncRNAs and 26,109 coding transcripts which collected from the most authoritative databases such as RefSeq, UCSC, Knowngenes, Ensembl and many related literatures can be detected by the microarray. Briefly, RNA purified from total RNA after removal of rRNA was amplified and transcribed into fluorescent cRNA and cDNA was labeled and hybridized to the Human LncRNA Array v2.0 (8660 K, Arraystar). 30,586 LncRNAs and 26,109coding transcripts which collected from the most authoritative databases such as RefSeq, UCSC Knowngenes, Ensembl and many related literatures can be detected by the microarray. The hybridized arrays were washed, fixed, and scanned using the Agilent DNA Microarray Scanner (G2505B) Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name diagnosis Experimental Factor Type diagnosis Comment[SecondaryAccession] GSE65263 Comment[GEOReleaseDate] 2015-12-31 Comment[ArrayExpressSubmissionDate] 2015-01-23 Comment[GEOLastUpdateDate] 2016-01-01 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE65263_normalized_data_with_probename.txt SDRF File E-GEOD-65263.sdrf.txt