Analysis of Gene Expression at the Functional Genomic Level Using Northern Blotting or PCR

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

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Validation of a Flour-Free Model Dough System for Throughput Studies of Baker's Yeast

Applied and Environmental Microbiology ◽

10.1128/aem.71.3.1142-1147.2005 ◽

2005 ◽

Vol 71 (3) ◽

pp. 1142-1147 ◽

Cited By ~ 28

Author(s):

Joaquin Panadero ◽

Francisca Randez-Gil ◽

Jose Antonio Prieto

Keyword(s):

Gene Expression ◽

Sugar Content ◽

Transcriptional Response ◽

Northern Blotting ◽

Baker’S Yeast ◽

Marker Genes ◽

Mrna Levels ◽

Baker's Yeast ◽

High Sugar ◽

High Sugar Content

ABSTRACT Evaluation of gene expression in baker's yeast requires the extraction and collection of pure samples of RNA. However, in bread dough this task is difficult due to the complex composition of the system. We found that a liquid model system can be used to analyze the transcriptional response of industrial strains in dough with a high sugar content. The production levels of CO2 and glycerol by two commercial strains in liquid and flour-based doughs were correlated. We extracted total RNA from both a liquid and a flour-based dough. We used Northern blotting to analyze mRNA levels of three stress marker genes, HSP26, GPD1, and ENA1, and 10 genes in different metabolic subcategories. All 13 genes had the same transcriptional profile in both systems. Hence, the model appears to effectively mimic the environment encountered by baker's yeast in high-sugar dough. The liquid dough can be used to help understand the connections between technological traits and biological functions and to facilitate studies of gene expression under commercially important, but experimentally intractable, conditions.

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Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation

Journal of Bacteriology ◽

10.1128/jb.00725-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5489-5498 ◽

Cited By ~ 41

Author(s):

Peng Gao ◽

Kenneth L. Pinkston ◽

Sreedhar R. Nallapareddy ◽

Ambro van Hoof ◽

Barbara E. Murray ◽

...

Keyword(s):

Gene Expression ◽

Enterococcus Faecalis ◽

Drug Targets ◽

Deletion Mutant ◽

Transcript Level ◽

Northern Blotting ◽

Gram Positive ◽

Gene Assay ◽

Functional Features ◽

Pilin Gene

ABSTRACT Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.

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An integrated reverse functional genomic and metabolic approach to understanding orotic acid-induced fatty liver

Physiological Genomics ◽

10.1152/physiolgenomics.00158.2003 ◽

2004 ◽

Vol 17 (2) ◽

pp. 140-149 ◽

Cited By ~ 78

Author(s):

Julian L. Griffin ◽

Stephanie A. Bonney ◽

Chris Mann ◽

Abdul M. Hebbachi ◽

Geoff F. Gibbons ◽

...

Keyword(s):

Gene Expression ◽

Fatty Liver ◽

Gene Expression Profiling ◽

Stress Responses ◽

Expression Profiling ◽

Orotic Acid ◽

Dna Microarrays ◽

Data Sets ◽

Functional Genomic ◽

H Nmr

In functional genomics, DNA microarrays for gene expression profiling are increasingly being used to provide insights into biological function or pathology. To better understand the significance of the multiple transcriptional changes across a time period, the temporal changes in phenotype must be described. Orotic acid-induced fatty liver disease was investigated at the transcriptional and metabolic levels using microarrays and metabolic profiling in two strains of rats. High-resolution 1H-NMR spectroscopic analysis of liver tissue indicated that Kyoto rats compared with Wistar rats are predisposed to the insult. Metabolite analysis and gene expression profiling following orotic acid treatment identified perturbed metabolic pathways, including those involved in fatty acid, triglyceride, and phospholipid synthesis, β-oxidation, altered nucleotide, methyl donor, and carbohydrate metabolism, and stress responses. Multivariate analysis and statistical bootstrapping were used to investigate co-responses with transcripts involved in metabolism and stress responses. This reverse functional genomic strategy highlighted the relationship between changes in the transcription of stearoyl-CoA desaturase 1 and those of other lipid-related transcripts with changes in NMR-derived lipid profiles. The results suggest that the integration of 1H-NMR and gene expression data sets represents a robust method for identifying a focused line of research in a complex system.

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Perforin gene expression in stimulated human peripheral blood T cells studied by in situ hybridization and Northern blotting analysis

Immunology Letters ◽

10.1016/0165-2478(92)90096-7 ◽

1992 ◽

Vol 33 (1) ◽

pp. 79-85

Author(s):

Chau-Ching Liu ◽

Shahin Rafii ◽

Hirotaka Koizumi ◽

Angela Granelli-Piperno ◽

John Ding-E Young

Keyword(s):

Gene Expression ◽

T Cells ◽

In Situ Hybridization ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Northern Blotting ◽

Blotting Analysis

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A genome-wide association study for nonalcoholic fatty liver disease identifies novel genetic loci and trait-relevant candidate genes in the Million Veteran Program

10.1101/2020.12.26.20248491 ◽

2021 ◽

Author(s):

Marijana Vujkovic ◽

Shweta Ramdas ◽

Kimberly M. Lorenz ◽

Carolin V. Schneider ◽

Joseph Park ◽

...

Keyword(s):

Gene Expression ◽

Liver Disease ◽

Fatty Liver ◽

Fatty Liver Disease ◽

Genome Wide Association Study ◽

Genome Wide Association ◽

Functional Genomic ◽

Nonalcoholic Fatty Liver ◽

Genomic Annotation ◽

Genome Wide

AbstractNonalcoholic fatty liver disease (NAFLD) is a prevalent, heritable trait that can progress to cancer and liver failure. Using our recently developed proxy definition for NAFLD based on chronic liver enzyme elevation without other causes of liver disease or alcohol misuse, we performed a multi-ancestry genome-wide association study in the Million Veteran Program with 90,408 NAFLD cases and 128,187 controls. Seventy-seven loci exceeded genome-wide significance of which 70 were novel, with an additional European-American specific and two African-American specific loci. Twelve of these loci were also significantly associated with quantitative hepatic fat on radiological imaging (n=44,289). Gene prioritization based on coding annotations, gene expression from GTEx, and functional genomic annotation identified candidate genes at 97% of loci. At eight loci, the allele associated with lower gene expression in liver was also associated with reduced risk of NAFLD, suggesting potential therapeutic relevance. Functional genomic annotation and gene-set enrichment demonstrated that associated loci were relevant to liver biology. We expand the catalog of genes influencing NAFLD, and provide a novel resource to understand its disease initiation, progression and therapy.

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Pairwise comparisons across species are problematic when analyzing functional genomic data

10.1101/107177 ◽

2017 ◽

Cited By ~ 4

Author(s):

Casey W. Dunn ◽

Felipe Zapata ◽

Catriona Munro ◽

Stefan Siebert ◽

Andreas Hejnol

Keyword(s):

Gene Expression ◽

Evolutionary Process ◽

Opportunity To Learn ◽

Genomic Data ◽

The Other ◽

Evolutionary Relationships ◽

Pairwise Comparisons ◽

Functional Genomic ◽

Functional Genomic Data ◽

Comparative Functional Genomics

AbstractThere is considerable interest in comparing functional genomic data across species. One goal of such work is to provide an integrated understanding of genome and phenotype evolution. Most comparative functional genomic studies have relied on multiple pairwise comparisons between species, an approach that does not incorporate information about the evolutionary relationships among species. The statistical problems that arise from not considering these relationships can lead pairwise approaches to the wrong conclusions, and are a missed opportunity to learn about biology that can only be understood in an explicit phylogenetic context. Here we examine two recently published studies that compare gene expression across species with pairwise methods, and find reason to question the original conclusions of both. One study interpreted pairwise comparisons of gene expression as support for the ortholog conjecture, the hypothesis that orthologs tend to be more similar than paralogs. The other study interpreted pairwise comparisons of embryonic gene expression across distantly related animals as evidence for a distinct evolutionary process that gave rise to phyla. In each study, distinct patterns of pairwise similarity among species were originally interpreted as evidence of particular evolutionary processes, but instead we find they reflect species relationships. These reanalyses concretely demonstrate the inadequacy of pairwise comparisons for analyzing functional genomic data across species. It will be critical to adopt phylogenetic comparative methods in future functional genomic work. Fortunately, phylogenetic comparative biology is also a rapidly advancing field with many methods that can be directly applied to functional genomic data.SignificanceComparisons of genome function between species are providing important insight into the evolutionary origins of diversity. Here we demonstrate that comparative functional genomics studies can come to the wrong conclusions if they do not take the relationships of species into account and instead rely on pairwise comparisons between species, as is common practice. We re-examined two previously published studies and found problems with pairwise comparisons that draw both their original conclusions into question. One study had found support for the ortholog conjecture and the other had concluded that the evolution of gene expression was different between animal phyla than within them. Our results demonstrate that to answer evolutionary questions about genome function, it is critical to consider evolutionary relationships.

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Pairwise comparisons across species are problematic when analyzing functional genomic data

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707515115 ◽

2018 ◽

Vol 115 (3) ◽

pp. E409-E417 ◽

Cited By ~ 40

Author(s):

Casey W. Dunn ◽

Felipe Zapata ◽

Catriona Munro ◽

Stefan Siebert ◽

Andreas Hejnol

Keyword(s):

Gene Expression ◽

Evolutionary Process ◽

Opportunity To Learn ◽

Genomic Data ◽

Pairwise Comparisons ◽

Functional Genomic ◽

Functional Genomic Data ◽

Genomic Studies ◽

Compare Gene Expression ◽

Missed Opportunity

There is considerable interest in comparing functional genomic data across species. One goal of such work is to provide an integrated understanding of genome and phenotype evolution. Most comparative functional genomic studies have relied on multiple pairwise comparisons between species, an approach that does not incorporate information about the evolutionary relationships among species. The statistical problems that arise from not considering these relationships can lead pairwise approaches to the wrong conclusions and are a missed opportunity to learn about biology that can only be understood in an explicit phylogenetic context. Here, we examine two recently published studies that compare gene expression across species with pairwise methods, and find reason to question the original conclusions of both. One study interpreted pairwise comparisons of gene expression as support for the ortholog conjecture, the hypothesis that orthologs tend to have more similar attributes (expression in this case) than paralogs. The other study interpreted pairwise comparisons of embryonic gene expression across distantly related animals as evidence for a distinct evolutionary process that gave rise to phyla. In each study, distinct patterns of pairwise similarity among species were originally interpreted as evidence of particular evolutionary processes, but instead, we find that they reflect species relationships. These reanalyses concretely show the inadequacy of pairwise comparisons for analyzing functional genomic data across species. It will be critical to adopt phylogenetic comparative methods in future functional genomic work. Fortunately, phylogenetic comparative biology is also a rapidly advancing field with many methods that can be directly applied to functional genomic data.

Download Full-text