scholarly journals MicroRNA expression profiling in bladder cancer: the challenge of next-generation sequencing in tissues and biofluids

2015 ◽  
Vol 138 (10) ◽  
pp. 2334-2345 ◽  
Author(s):  
Giuseppe Matullo ◽  
Alessio Naccarati ◽  
Barbara Pardini
Keyword(s):  
Bladder Cancer ◽  
Next Generation Sequencing ◽  
Expression Profiling ◽  
Microrna Expression ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals New methods for next generation sequencing based microRNA expression profiling

BMC Genomics ◽  
2010 ◽  
Vol 11 (1) ◽  
pp. 716 ◽  
Author(s):  
Henk PJ Buermans ◽  
Yavuz Ariyurek ◽  
Gertjan van Ommen ◽  
Johan T den Dunnen ◽  
Peter AC 't Hoen
Keyword(s):  
Next Generation Sequencing ◽  
Expression Profiling ◽  
Microrna Expression ◽  
Next Generation ◽  
New Methods ◽  
Generation Sequencing

243 GENE EXPRESSION PROFILING BY NEXT-GENERATION SEQUENCING OF cDNA SAMPLES FROM INNER CELL MASS AND TROPHECTODERM OF A SINGLE-DAY 12 PORCINE EMBRYO

2010 ◽  
Vol 22 (1) ◽  
pp. 279
Author(s):  
S. C. Isom ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Cdna Fragments ◽  
Generation Sequencing

Traditional microarray approaches to gene expression profiling often require RNA or cDNA amplification when working with extremely small or valuable tissue samples.This process is generally viewed as being undesirable because there is potential for bias to be introduced during amplification. Very recently, the so-called next-generation sequencing technologies were adapted for use in global gene expression profiling. Herein we report our efforts to apply these sequencing technologies to assess relative transcript abundances in pre-implantation-stage porcine embryos, without additional nucleic acid amplification before sequencing. As a proof-of-principle experiment, we have isolated total RNA from the embryonic disc (inner cell mass; ICM) and a small piece of trophectoderm (TE) from a Day 12 in vivo-produced embryo, which were estimated to be composed of 500 to 1000 cells each. The RNA was reverse transcribed using oligo-dT priming followed by second-strand cDNA synthesis. The double-stranded cDNA was then randomly sheared by sonication, and 10 ng of double-stranded cDNA fragments was used for sample preparation before sequencing. Prepared cDNA fragments (at 7 picomolar concentrations) were submitted for sequencing using the Illumina/Solexa platform as recommended. The millions of short (36 bp) reads generated by Illumina sequencing for each sample were then aligned to the swine UniGene database from NCBI, allowing for zero or one mismatches. Relative transcript abundances between cell types were profiled by considering the read counts for a given UniGene member as a percentage of the total number of reads generated for each cell type. It was demonstrated that approximately 11 000 and 9000 UniGene members were represented by a normalized average of 5 or more short reads per lane (0.001% of the total) in the ICM and TE samples, respectively. As expected, pluripotency factors, chromatin remodeling components, and cell-cell communication molecules were overrepresented in the ICM sample as compared with the TE sample. Conversely, epithelial determinants, ion transporters, and components of the steroid biosynthesis pathways were more abundant in the TE sample than in the ICM sample. Relative abundances of representative transcripts in these samples were verified by quantitative RT-PCR. In conclusion, we demonstrate the utility of next-generation sequencing technologies for gene expression profiling using even minute tissue samples and show that such analyses are possible even in species without a sequenced genome.

Seasonal variation of urinary microRNA expression in male goats (Capra hircus) as assessed by next generation sequencing

2014 ◽  
Vol 199 ◽  
pp. 1-15 ◽  
Author(s):  
Kristy M. Longpre ◽  
Noah S. Kinstlinger ◽  
Edward A. Mead ◽  
Yongping Wang ◽  
Austin P. Thekkumthala ◽  
...  
Keyword(s):  
Seasonal Variation ◽  
Next Generation Sequencing ◽  
Microrna Expression ◽  
Capra Hircus ◽  
Next Generation ◽  
Generation Sequencing

RNAseq in addition to next generation sequencing in advanced genitourinary cancers reveals transcriptomic silencing of DNA mutations: Implications for resistance to targeted therapeutics.

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 583-583 ◽  
Author(s):  
Jacob J. Adashek ◽  
Shumei Kato ◽  
Rahul Parulkar ◽  
Christopher Szeto ◽  
Sandeep K. Reddy ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bladder Cancer ◽  
Next Generation Sequencing ◽  
Kidney Cancer ◽  
Next Generation ◽  
Dna And Rna ◽  
Number Of Patients ◽  
Study Results ◽  
Dna Alterations ◽  
Generation Sequencing

583 Background: Next generation sequencing (NGS) for advanced tumors is becoming more routine. However, not all patients respond to precision matched treatments. We hypothesized that one potential reason for treatment failure with targeted therapy could be discrepancies between DNA alterations and RNA expression. Methods: Tumor samples from patients with metastatic kidney, bladder, and prostate cancer were analyzed by whole exome or whole genome NGS and RNA sequencing (CLIA-certified laboratory; NantOmics LLC, Santa Cruz, CA). Only known pathogenic driver alterations were analyzed in the current study. Results: Of 45 patients, 10 had kidney cancer, 18 had bladder cancer; and 17 had prostate cancer. Median age was 66 years (range, 28 - 86). The most commonly altered genes were TP53 (35.6% [16/45]), PIK3CA (15.6% [7/45]), FGFR3 (11.1% [5/45]), ALK (8.9% [4/45]), and ATM (8.9% [4/45]). In total, 86 pathogenic DNA alterations were identified; 17 of these (19.8%) were not observed at the RNA level. Among 45 patients, 31.1% (14/45) had ≥1 DNA alteration that was not expressed at the RNA level. Discordance between DNA and RNA was seen in 40% of patients with kidney cancer (4/10), 28% of patients with bladder cancer (5/18), and 29% with prostate cancer (5/17). Examples of genes that had pathogenic DNA alterations not seen at the RNA level included ALK (four discordant cases), KDR (three discordant cases) and GNAS (one discordant case). On the other hand, alterations involving certain genes showed 100% concordance between DNA and RNA: TP53 [N = 16], PIK3CA [N = 7], and FGFR3 [N = 5]). Conclusions: A significant number of patients with genitourinary tumors had DNA alterations that are silenced at the RNA level (19.8%). Transcriptomic silencing merits additional investigation as a mechanism that could mediate resistance to therapeutics targeted at cognate alterations.

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