Next Generation Sequencing Facilitates Quantitative Analysis of Ramos and Rituximab-resistant Ramos cell Transcriptomes
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE112592 | GEO | 2019/02/13
REPOSITORIES: GEO
Similar Datasets
Project description:Next Generation Sequencing Facilitates Quantitative Analysis of Ramos and Rituximab-resistant Ramos cell Transcriptomes
| PRJNA448419 | ENA
Project description:PurposeNext-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods and to evaluate protocols for optimal high-throughput data analysis.MethodsRetinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl(-/-)) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows-Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT-PCR validation was performed using TaqMan and SYBR Green assays.ResultsUsing an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl(-/-) mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT-PCR. RNA-seq data had a linear relationship with qRT-PCR for more than four orders of magnitude and a goodness of fit (R(2)) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl(-/-) retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT-PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.ConclusionsOur study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
| S-EPMC3233386 | biostudies-literature
Project description:Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived normal human kidney transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis The kidney tissue was immediately placed and stored in RNAlater® (Ambion), according to the manufacturer’s instruction. The tissue was manually microdissected under microscope in RNAlater® pool for glomerular and tubular compartment. Dissected tissue was homogenized and RNA was prepared using RNAeasy mini columns (Qiagen, Valencia, CA, US), according to the manufacturer’s instructions. RNA quality and quantity were determined using the Laboratory-on-Chip Total RNA PicoKit Agilent BioAnalyzer. Only samples without evidence of degradation were further used (RNA Integrity Number >6).
2014-08-06 | E-GEOD-60119 | biostudies-arrayexpress
Project description:We used RNAseq to analyse the transcriptomes of cycling and quiescent livers from cTKO (Rblox/lox;p130lox/lox;p107?/?) and control mice. mRNA profiles of cycling and quiescent control and TKO liver tissue were generated by deep sequencing in duplicate
2014-07-12 | E-GEOD-43631 | biostudies-arrayexpress
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods: Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl-/-) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Retinal mRNA profiles of 21-day old wild type (WT) and Nrl-/- mice were generated by deep sequencing, in triplicate, using Illumina GAIIx.
2011-10-25 | E-GEOD-33141 | biostudies-arrayexpress
Project description:Analysis of gene expression level. The hypothesis tested in the present study was that fl4 mutant influence the ER stress pathway. Results provide important information of the gene expression level regulation of different ER stress pathways and apoptosis Endosperm mRNA profiles of 19DAP wild type (WT) and fl4 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2000 (Zea mays).
2014-01-18 | E-GEOD-54184 | biostudies-arrayexpress
Project description:Analysis of gene expression level. The hypothesis tested in the present study was that pro1 mutant influence the cell cycle pathway. Results provide important information of the cell cycle-related gene expression level regulation of cell cycle transition from G1 to S phase. Seedling mRNA profiles of 14DAG wild type (WT) and pro1 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2000 (Zea mays).
2015-03-31 | E-GEOD-56004 | biostudies-arrayexpress
Project description:Analysis of gene expression level. The hypothesis tested in the present study was that opaque2 mutant influence the expression of storage proteins. Results provide important information of the gene expression level regulation of storage proteins and other biological processes. Endosperm mRNA profiles of 15DAP wild type (WT) and opaque2 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 (Zea mays).
2014-10-15 | E-GEOD-61830 | biostudies-arrayexpress
Project description:Analysis of gene expression level. The hypothesis tested in the present study was that rea1 mutant influence the expression of translation and nucleosome assembly associated genes. Results provide important information of the gene expression level regulation of ribosome proteins, elongation factors, histones and genes associated with other biological processes. Endosperm mRNA profiles of 15DAP wild type (WT) and rea1 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 (Zea mays).
2015-04-25 | E-GEOD-67080 | biostudies-arrayexpress
Project description:Analysis of gene expression level. The hypothesis tested in the present study was that dek35 mutant influence the expression of mitochondrial oxidative phosphorylation associated genes. Results provide important information of the gene expression level regulation of mitochondrial complexes assembly proteins, translational elongation factors and genes associated with other biological processes. Endosperm mRNA profiles of 18DAP wild type (WT) and dek35 mutant were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 (Zea mays).
2016-03-25 | E-GEOD-79583 | biostudies-arrayexpress