Whole transcriptome analysis with sequencing: methods, challenges and potential solutions

Jiang, Zhihua; Zhou, Xiang; Li, Rui; Michal, Jennifer J.; Zhang, Shuwen; Dodson, Michael V.; Zhang, Zhiwu; Harland, Richard M.

doi:10.1007/s00018-015-1934-y

Cited by 179 publications

(124 citation statements)

References 97 publications

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“…The Sequenom MassARRAY system, based on MALDI-TOF mass spectrometry, was originally designed to analyze SNPs in amplified DNA fragments but was then adapted in molecular oncology for detection of known well-defined mutations (single-nucleotide variants, SNV) [25,26]. The principle in this application is that mutant and germline alleles for a given point mutation produce single-allele base extension reaction products of different masses that are then resolved by MALDI-TOF Mass Spectrometry [26].…”

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

“…The principle in this application is that mutant and germline alleles for a given point mutation produce single-allele base extension reaction products of different masses that are then resolved by MALDI-TOF Mass Spectrometry [26]. The assay consists of an initial locus-specific PCR reaction, followed by single base extension using mass-modified dideoxynucleotide terminators of an oligonucleotide primer which anneals immediately upstream of the polymorphic site of interest [26]. The Sequenom platform uses matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to distinguish the products of primer extension reactions (performed on PCR products) in a sequence-specific manner based on the mass and charge [25,26].…”

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

“…The assay consists of an initial locus-specific PCR reaction, followed by single base extension using mass-modified dideoxynucleotide terminators of an oligonucleotide primer which anneals immediately upstream of the polymorphic site of interest [26]. The Sequenom platform uses matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to distinguish the products of primer extension reactions (performed on PCR products) in a sequence-specific manner based on the mass and charge [25,26]. Using MALDI-TOF mass spectrometry, the distinct mass of the extended primer identifies the SNP allele or the somatic single nucleotide variant [25,26].…”

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

“…The Sequenom platform uses matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to distinguish the products of primer extension reactions (performed on PCR products) in a sequence-specific manner based on the mass and charge [25,26]. Using MALDI-TOF mass spectrometry, the distinct mass of the extended primer identifies the SNP allele or the somatic single nucleotide variant [25,26]. In our experience, MALDITOF-MS is a very powerful platform; the automated workflow and relatively Short hands on time multiplexing of multiple samples on one array chip with a total time of 9.5 hours from PCR setup to report generation.…”

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

See 4 more Smart Citations

The history of dermatology, venereology and dermatopathology in different countries - China

Zhang¹,

Chen²,

Zhang³

et al. 2016

Glob Dermatol

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Volume 3(4): [352][353][354][355][356][357][358] hybridization (FISH), comparative genomic hybridization and SNP array (CGH+SNP) array, and gene expression signature (quantitative reverse transcriptase PCR). Platforms for therapeutic guidance include Sanger sequencing, matrix-associated laser di-ionization time of flight mass spectrometry (MALDITOF-MS), and high-throughput targeted next-generation sequencing (NGS). Each of these platform categories is discussed below. Fluorescence in situ hybridizationConventional cytogenetics and CGH paved the way for FISH in dermatopathology. The initial data published by Bastian et al. in 2002 showed recurrent genomic aberrations in melanomas involving chromosomes 9, 10, 7, and 6, in contrast to most Spitz nevi, which do not show aberrations [3]. The following years witnessed a plethora of publications addressing the application of FISH and its diagnostic and prognostic value in melanocytic lesions.FISH uses fluorescent-labeled probes targeting specific genomic areas of interest. Hybridization of these probes is performed on baked IntroductionSince the sequencing of nearly the entire human genome was first accomplished in 2003 the world of molecular pathology has experienced an exponential evolution [1]. This process evolved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH), examining hundreds of base pairs and identifying singlenucleotide polymorphisms (SNP), and next-generation sequencing (providing single base pair resolution) [1]. Whole genome nextgeneration sequencing remains a cost-prohibitive method for many investigators, although considerable effort has been made to lower its cost for better incorporation into clinical practice. With the advent of newer technologies the cost of aCGH decreased recent years and the resolution of genome mapping increased [2]. A similar cost reduction has been seen in targeted next-generation sequencing, which has gained tremendous popularity and become the sine qua non for standard testing in personalized medicine centers.Understanding the specific needs of the patient population in each practice and tailoring the different available platforms in that regard, remains the most important point that needs to be taken in consideration when implementing next-generation sequencing techniques. In this review, we will focus on clinical platforms that have shown promising results in diagnosis, prognosis, and theranostics in the clinical practice in dermatopathology. Data generated by these methods will enable pathologists and clinicians to better understand the numerous challenging cases and to design treatment plans customized to the patient's specific tumor. We will not discuss available techniques that are currently limited to research use (whole-genome sequencing, non-targeted RNA sequencing).From a practical standpoint, the different molecular platforms in clinical dermato...

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Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

Section: Matrix Associated Laser Di-ionization Time Of Flight Mass Spmentioning

confidence: 99%

See 3 more Smart Citations

The history of dermatology, venereology and dermatopathology in different countries - China

Zhang¹,

Chen²,

Zhang³

et al. 2016

Glob Dermatol

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The proliferation role of LH on porcine primordial germ cell‐like cells (pPGCLCs) through ceRNA network construction

Zhang

Tian

Zhang

et al. 2021

Clinical & Translational Med

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Background The transdifferentiation of skin‐derived stem cells (SDSCs) into primordial germ cell‐like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells research in recent years. This technology provides a new theoretical basis for the treatment of human infertility. However, the transdifferentiation efficiency of SDSCs to PGCLCs is very low, and scientists are still exploring ways to improve this efficiency or promote the proliferation of PGCLCs. This study aims to investigate the molecular mechanism of luteinising hormone (LH) to enhance porcine PGCLCs (pPGCLCs) proliferation. Results In this study, we dissected the proliferation regulatory network of pPGCLCs by whole transcriptome sequencing, and the results showed that the pituitary‐secreted reproductive hormone LH significantly promoted the proliferation of pPGCLCs. We combined whole transcriptome sequencing and related validation experiments to explore the mechanism of LH on the proliferation of pPGCLCs, and found that LH could affect the expression of Hippo signalling pathway‐related mRNAs, miRNAs and lncRNAs in pPGCLCs. Conclusions For the first time, we found that LH promotes pPGCLCs proliferation through the competing endogenous RNA (ceRNA) regulatory networks and Hippo signalling pathway. This finding may help to elucidate the molecular mechanism by which LH promotes pPGCLCs proliferation.

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Transcriptomic identification of differentially expressed genes in Levonorgestrel resistant endometrial cancer cell lines

Dore

Faya

Filoche

et al. 2023

Molecular Carcinogenesis

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Endometrial cancer (EC) is the most common gynecologic malignancy in the world and incidence is steadily increasing. The Levonorgestrel Intrauterine System (LNG‐IUS) is an alternative conservative treatment for early‐stage EC, however, Levonorgestrel (LNG) resistance occurs for 1 in 3 people. This study aimed to present potential LNG resistance mechanisms and identify differentially expressed genes (DEGs) in EC cell lines. Two LNG resistant cell lines were developed through long term culture in LNG (MFE296R and MFE319R). Whole transcriptome sequencing was carried out on triplicate RNA samples. EdgeR v3.32.1 was used to identify differentially DEGs. Blast2go V6.0 (BioBam software) was used for functional annotation and analysis of genomic datasets. Protein interactions were investigated using the STRING database, including the identification of genes with high levels of interaction (HUB genes). Select DEGs and HUB genes were validated by quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blot. Fifteen DEGs were identified according to FDR < 0.05 and logFC < 2. Protein analysis identified six HUB genes with a degree of connectivity > 10. Relative mRNA expression of MAOA, MAOB, THRSP, CD80, NDP, LINC01474, DUSP2 and CXCL8 was significantly upregulated in both LNGR cell lines. Relative protein expression of GNAO1 and MAOA were significantly upregulated in both LNGR cell lines. This research identified novel markers of resistance in LNGR cell lines. We discussed potential mechanisms of LNG resistance including dedifferentiation and immunostimulation. The next step for this research is to validate these findings further in both translational and clinical settings.

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Whole transcriptome analysis with sequencing: methods, challenges and potential solutions

Cited by 179 publications

References 97 publications

The history of dermatology, venereology and dermatopathology in different countries - China

The history of dermatology, venereology and dermatopathology in different countries - China

The proliferation role of LH on porcine primordial germ cell‐like cells (pPGCLCs) through ceRNA network construction

Transcriptomic identification of differentially expressed genes in Levonorgestrel resistant endometrial cancer cell lines

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