Dataset Information

MiR-22 promotes stem cell traits via activating WNT/β-catenin signaling in cSCC

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized system-based cell pathway analysis. The purposes of this study were to compare and analyze the expression differences of skin squamous cell line A431 in stem cells after Mir-22 deletion. Methods: mRNA profiles of control and case cells were generated by deep sequencing, in duplicate, using Illumina NovaSeq 6000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods:Bowtie2 to map clean reads to reference gene and use HISAT to reference genome.Bowtie2 parameters forSE reads:-q--phred64--sensitive--dpad0--gbar and HISTAparameters forSE reads:-p8--phred64--sensitive-I1-X 1000. qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 24 million sequence reads per sample to the human genome (build hg19) and identified 17000 transcripts in control and case cells with RSEM workflow. Approximately 5% of the transcripts showed differential expression between control and case cells, with a fold change ≥1.5 and p value <0.01. Altered expression of 10 candidate genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method.

ORGANISM(S): Homo sapiens

PROVIDER: GSE156255 | GEO | 2020/08/15

REPOSITORIES: GEO

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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 thyroid cancer cell line k1 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: mRNA profiles of shCtrl and shTBX3 cells were generated by deep sequencing, in duplicate, using BGISEQ-500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods:Bowtie2 to map clean reads to reference gene and use HISAT to reference genome.Bowtie2 parameters forSE reads:-q--phred64--sensitive--dpad0--gbar and HISTAparameters forSE reads:-p8--phred64--sensitive-I1-X 1000. qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 24 million sequence reads per sample to the human genome (build hg19) and identified 20511 transcripts in K1-shCtrl and K1-shTBX3 cells with RSEM workflow. Approximately 5% of the transcripts showed differential expression between K1-shCtrl and K1-shTBX3 cells, with a fold change ≥1.5 and p value <0.01. Altered expression of 16 candidate 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 thyroid cancer function. Conclusions: Our study represents the first detailed analysis of thyroid cancer cell line k1 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.

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 WT and dKO round spermatids transcriptome profiling (RNA-seq) Methods: Adult WT and dKO round spermatids mRNA profiles mice were generated by deep sequencing, in dulplicate. 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 dKO round spermatids, 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. Adult wild type (WT) and dKO mouse round spermatids were generated by deep sequencing, in dulplicate, using Illumina GAIIx.

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.

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MiR-22 promotes stem cell traits via activating WNT/β-catenin signaling in cSCC

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