Multiple Gene Expression Analysis Research Articles

The role of hydrogen-rich water in delaying the pulp breakdown of litchi fruit during postharvest storage

Previous studies have indicated that hydrogen-rich water (HW) treatment can delay fruit ripening and senescence. However, little is known about the HW-delaying pulp breakdown. In this study, eight physiological characteristics revealed that HW treatment delayed both pericarp browning and pulp breakdown of litchi fruit. To gain a comprehensive understanding of the changes in litchi pulp, a combination of multiple metabolomics and gene expression analyses was conducted, assessing 67 primary metabolites, 103 volatiles, 31 amino acids, and 13 crucial metabolite-related genes. Results showed that HW treatment promoted starch degradation, decelerated cell wall degradation and glycolysis, and maintained the flavor and quality of litchi fruit. Furthermore, HW treatment stimulated the production of volatile alcohols, aldehydes, ketones, olefins, and amino acids, which might play a vital role in HW-delaying pulp breakdown. This study sheds light on the mechanism by which HW delayed pulp breakdown by investigating small molecule metabolites and metabolic pathways.

GOTermViewer: Visualization of Gene Ontology Enrichment in Multiple Differential Gene Expression Analyses.

Gene ontology phrases are a widely used set of hierarchical terms that describe the biological properties of genes. These terms are then used to annotate individual genes, making it possible to determine the likely physiological properties of groups of genes such as a list of differentially expressed genes. Consequently, their ability to predict changes in biological features and functions based on alterations in gene expression has made gene ontology terms popular in the wide range of bioinformatic fields, such as differential gene expression and evolutionary biology. However, while they make the analysis easier, it is seldom easy to convey the results in a readily understandable manner. A number of applications have been developed to visualize gene ontology (GO) term enrichment; however, these solutions tend to focus on the display of aggregated results from a single analysis, making them unsuitable for the analysis of a series of experiments such as a time course or response to different drug treatments. As multiple pair wise comparisons are becoming a common feature of RNA profiling experiments, the absence of a mechanism to easily compare them is a significant problem. Consequently, to overcome this obstacle, we have developed GOTermViewer, an application that displays GO term enrichment data as determined by GOstats such that changes in physiological response across a number of individual analyses across a time course or range of drug treatments can be visualized.

Activatable Fluorescence-Encoded Nanoprobes Enable Simple Multiplexed RNA Imaging in Live Cells.

Benefiting from superior programmable performance and flexible design of DNA technologies, a variety of single-molecule RNA fluorescence imaging methodologies have been reported. However, the multiplexing capability is restricted owing to the spectral overlap of fluorophores. To overcome this limitation, some inspiring multiplex imaging strategies have been developed, but in practice, it remains challenging to achieve convenient and rapid imaging in live cells due to complex designs and additional pretreatments to increase cell permeability. Here, we report an activatable fluorescence-encoded nanoprobe (AFENP) strategy, through which fluorescence-encoded functional modules for qualitative analysis and activated nucleic acid assemblies functional modules for quantitative testing enable simple multiplexed RNA imaging in single live cells. As a proof of principle, by two distinguishable fluorophores (fluorescein and rhodamine B) and their seven distinctly differentiated intensity levels, self-assembled AFENP enables simplified and quick simultaneous in situ detection and imaging of seven types of targets in live single cells because the fluorescent quantitative signal is activated only in the presence of target avoiding the washing procedures and additional pretreatment to increase cell permeability is undesired. We expect that this practical single-cell analysis platform will be adopted for multiple gene expression analysis and imaging in live cells on account of its simplicity and multiplex capability.

Integration of Single-Cell Transcriptomics With a High Throughput Functional Screening Assay to Resolve Cell Type, Growth Kinetics, and Stemness Heterogeneity Within the Comma-1D Cell Line.

Cell lines are one of the most frequently implemented model systems in life sciences research as they provide reproducible high throughput testing. Differentiation of cell cultures varies by line and, in some cases, can result in functional modifications within a population. Although research is increasingly dependent on these in vitro model systems, the heterogeneity within cell lines has not been thoroughly investigated. Here, we have leveraged high throughput single-cell assays to investigate the Comma-1D mouse cell line that is known to differentiate in culture. Using scRNASeq and custom single-cell phenotype assays, we resolve the clonal heterogeneity within the referenced cell line on the genomic and functional level. We performed a cohesive analysis of the transcriptome of 5,195 sequenced cells, of which 85.3% of the total reads successfully mapped to the mm10-3.0.0 reference genome. Across multiple gene expression analysis pipelines, both luminal and myoepithelial lineages were observed. Deep differential gene expression analysis revealed eight subclusters identified as luminal progenitor, luminal differentiated, myoepithelial differentiated, and fibroblast subpopulations—suggesting functional clustering within each lineage. Gene expression of published mammary stem cell (MaSC) markers Epcam, Cd49f, and Sca-1 was detected across the population, with 116 (2.23%) sequenced cells expressing all three markers. To gain insight into functional heterogeneity, cells with patterned MaSC marker expression were isolated and phenotypically investigated through a custom single-cell high throughput assay. The comparison of growth kinetics demonstrates functional heterogeneity within each cell cluster while also illustrating significant limitations in current cell isolation methods. We outlined the upstream use of our novel automated cell identification platform—to be used prior to single-cell culture—for reduced cell stress and improved rare cell identification and capture. Through compounding single-cell pipelines, we better reveal the heterogeneity within Comma-1D to identify subpopulations with specific functional characteristics.

DNMT1 Expression and DNA Methylation in Intraductal Papillary Neoplasms of the Bile Duct.

Intraductal papillary neoplasms of the bile duct (IPNB) are histologically and clinically classified as type 1 and 2. This study aimed to identify the differences between these two types. Based on multiple gene expression analysis (MGEA) using type 1, type 2, and pancreatic intraductal papillary mucinous neoplasms (n=4, 6, and 5, respectively), immunohistochemistry of DNMT1 and methylation-specific PCR for p16, APC, BRCA1, hMLH1, TIMP3, and SOX17 were performed on type 1 and 2 IPNBs (n=14, each). The DNMT1 protein was highly expressed (p<0.001) in 28.6% of type 1 cases and all type 2 cases. The DNA methylation ratio for the six genes in total as well as for SOX17 was lower in type 1 than in type 2 (p<0.05 each). Type 2 IPNB showed increased DNMT1 protein expression and increased DNA methylation frequency of the examined tumor suppressor genes compared to type 1. DNMT1 IHC may be helpful in discriminating between these two types.

Upregulation of follistatin and low apoptotic activity in intraductal oncocytic papillary neoplasm of the pancreatobiliary system

Intraductal oncocytic papillary neoplasm (IOPN) is a rare intraductal tumor of the pancreatobiliary system. Currently, little is known about its distinct characteristics, unlike intraductal papillary mucinous neoplasms (IPMN) and intraductal papillary neoplasms of the bile duct (IPNB). The present study compared 22 IOPNs (18 pancreatic and 4 biliary) with those of 61 IPMNs/8 IPNBs. IOPNs were classified into pure and combined types, depending on the coexistence of IPMN/IPNB. Multiple gene expression analysis (nCounter system) was performed, and hierarchical clustering analysis separated IOPNs(n = 4) and IPMNs(n = 3)/ IPNBs(n = 3), and pathway score analysis supported the result. Volcano plot identified follistatin (FST) as the most upregulated mRNA in IOPN in comparison to the gastric subtype (log2 fold change of 5.34) and the intestinal subtype (that of 5.81) of IPMN/IPNB. The expression of FST in IOPN was also high in quantitative polymerase chain reaction and immunohistochemical analysis. We also found lower apoptotic activity in IOPN, particularly in pure type, compared to high-grade or invasive IPMN/IPNB using immunohistochemistry for cleaved caspase 3. But, combined type IOPN was more similar to IPMN/IPNB than pure IOPN. In conclusion, we proved that IOPN, particularly pure IOPN, is distinct from IPMN/IPNB in FST mRNA overexpression and exhibits lower apoptotic activity.

Identification of RING-H2 Gene Candidates Related to Wood Formation in Poplar

RING-H2 genes, the most abundant RING-type genes encoding putative ubiquitin ligase E3, are involved in diverse biological processes. Whether RING-H2 genes are related to wood formation remains to be identified in trees. In this study, we identified 288 RING-H2 genes in Populus trichocarpa, and found that the segmental and tandem duplication events contributed to RING-H2 gene expansion. Microarray dataset (from Affymetrix poplar genome arrays) showed that 64 of the 249 RING-H2 genes were highly or preferentially expressed in stem xylem. According to the AspWood RNAseq dataset, the transcription levels of genes PtrRHH21, 33, 48, 69, 88, 93, 94, 121, 141, 166, 175, 192, 208, 214, 250 and 257 were significantly increased in the xylem ranging from the expanding xylem to the lignifying xylem, suggesting their association with wood formation. Promoter analyses revealed that most of the preferentially xylem-expressed RING-H2 genes possessed SNBE, TERE, M46RE, AC and SMRE cis-elements, which are involved in secondary cell wall biosynthesis and programmed cell death. Based on the promoter GUS-based analysis result, PtrRHH94 was indicated to be associated with wood formation in transgenic P. trichocarpa. Taken together, dozens of Populus RING-H2 gene candidates associated with wood formation have been identified based on multiple gene expression analyses.

Identification of aberrantly methylated differentially expressed genes in glioblastoma multiforme and their association with patient survival.

Glioblastoma multiforme (GBM) is the most malignant primary tumour type of the central nervous system with limited therapeutic options and poor prognosis, and its pathogenic mechanisms have remained to be fully elucidated. Aberrant DNA methylation is involved in multiple biological processes and may contribute to the occurrence and development of GBM by affecting the expression of certain genes. However, the specific molecular mechanisms remain to be fully elucidated. The present study focused on uncovering differentially expressed genes with altered methylation status in GBM and aimed to discover novel biomarkers for the diagnosis and treatment of GBM. These genes were identified by combined analysis of multiple gene expression and methylation datasets from gene expression omnibus (GSE16011, GSE50161 and GSE 50923) to increase the reliability. In addition, The Cancer Genome Atlas (TCGA) dataset for GBM was used to test the stability of the results. Overall, 251 hypomethylated upregulated genes (Hypo-UGs) and 199 hypermethylated downregulated genes (Hyper-DGs) were identified in the present study. Functional enrichment analysis revealed that the Hypo-UGs are involved in the regulation of immune- and infection-associated signalling, while the Hyper-DGs are involved in the regulation of synaptic transmission. The three hub genes for Hyper-DGs (somatostatin, neuropeptide Y and adenylate cyclase 2) and five hub genes for Hypo-UGs [interleukin-8, matrix metalloproteinase (MMP)9, cyclin-dependent kinase 1, 2′-5′-oligoadenylate synthetase 1, C-X-C motif chemokine ligand 10 and MMP2] were identified by protein-protein interaction network analysis. Among the Hypo-UGs and Hyper-DGs, overexpression of C-type lectin domain containing 5A, epithelial membrane protein 3, solute carrier family 43 member 3, STEAP3 metalloreductase, tumour necrosis factor α-induced protein 6 and apolipoprotein B mRNA editing enzyme catalytic subunit 3G was significantly associated with poor prognosis in the TCGA and GSE16011 datasets (P<0.001). In conclusion, the present study uncovered numerous novel aberrantly methylated genes and pathways associated with GBM. Methylation-based markers, including the hub genes and prognostic genes identified, may potentially serve as markers for the diagnosis of GBM and targets for its treatment.

Multiple gene expression profiling suggests epithelial dysfunction in polypoid chronic rhinosinusitis.

SUMMARYChronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder resulting from a complex gene-environment interaction. Although its aetiology remains elusive, numerous studies reported gene expression alterations of factors apparently implicated in all aspects of the inflammatory response. However, most investigations are limited, unconfirmed analyses of a single gene. Moreover, studies concerning multiple gene expression analyses, usually on inflammatory mediators (e.g. cytokines), show contrasting outcomes in part due to use of heterogeneous samples or methodologies with limited power. In this scenario, our goal was to simultaneously evaluate the expression of a panel of selected genes (AQP5, MUC5AC, CAV1, LTF, COX2, PGDS, TNFα, TGFβ1, MGB1) potentially involved in CRS inflammatory mechanisms. While most of the samples collected were excluded from the analysis because of poor quality RNA, we were able to demonstrate statistically significant downregulation of the AQP5, CAV1, LTF, MGB1 genes in a specific subset of polypoid CRS (patients without typical comorbidities), which might suggest relevant underlying epithelial dysfunction. Further studies are needed to enrich our knowledge on the pathogenesis of CRS. Forthcoming approaches might utilise next-generation RNA sequencing and comprehensive bioinformatics analyses to better characterise the transcriptome profiles of CRS endotypes.

Notch pathway connections in primary leukaemia samples of limited size

BackgroundThe Notch pathway combined with other signalling molecules acts specifically for the development of each blood cell type and differentiation stage. A causative role of Notch dysfunction in leukaemia development has been found in many studies so, initially only for T- acute lymphoblastic leukaemia (T-ALL) but more recently also for B cell and myeloid leukaemia. The aim of our study is to introduce a method for multiple direct analysis of the Notch pathway partners in a population of only 500 or fewer cells. The notion of this method consists in gaining insight into gene expression at the level of the malignant clone population. A small number of cells is a significant limitation when working on primary cells either when freshly isolated or when analysed after several days in cocultures.MethodsThe primers were designed to avoid genomic amplification through the selection of 3′ and 5′ primers that hybridise with different exons. Cell lines and primary cells were collected and multiplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) performed on a descending number of cells, ranging from 2,500 cells up to 50 cells per sample, for the Notch pathway genes and other transcription factors important for cell differentiation. ImageJ program, STATISTICA 13.1 software package and Student’s t-test were used for statistical evaluation. We checked protein expression by western blot.ResultsWe characterised the gene expression levels of Notch, Ikaros and Parp genes in leukaemia cell lines of B and T origin and in primary leukaemia samples of limited size. We further compared our results to the cDNA analysis obtained by total RNA isolation from a large number of cells as routinely performed in clinical laboratories, and finally tested the method described on primary cells from leukaemia patients.ConclusionsThis rapid multiple gene expression analysis of a small population of cells provides efficient cell classification determining malignant changes as an important additional information for clinical leukaemia diagnostics as well as for in vitro studies of primary cells.

BcGST-an interactive bias-correction method to identify over-represented gene-sets in boutique arrays.

Gene annotation and pathway databases such as Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes are important tools in Gene-Set Test (GST) that describe gene biological functions and associated pathways. GST aims to establish an association relationship between a gene-set of interest and an annotation. Importantly, GST tests for over-representation of genes in an annotation term. One implicit assumption of GST is that the gene expression platform captures the complete or a very large proportion of the genome. However, this assumption is neither satisfied for the increasingly popular boutique array nor the custom designed gene expression profiling platform. Specifically, conventional GST is no longer appropriate due to the gene-set selection bias induced during the construction of these platforms. We propose bcGST, a bias-corrected GST by introducing bias-correction terms in the contingency table needed for calculating the Fisher's Exact Test. The adjustment method works by estimating the proportion of genes captured on the array with respect to the genome in order to assist filtration of annotation terms that would otherwise be falsely included or excluded. We illustrate the practicality of bcGST and its stability through multiple differential gene expression analyses in melanoma and the Cancer Genome Atlas cancer studies. The bcGST method is made available as a Shiny web application at http://shiny.maths.usyd.edu.au/bcGST/. Supplementary data are available at Bioinformatics online.

Evaluation of cervical ripening based on multiple gene expression analysis and its predictability for onset of labor

Evaluation of cervical ripening based on multiple gene expression analysis and its predictability for onset of labor

Multiplexed optical coding nanobeads and their application in single-molecule counting analysis for multiple gene expression analysis

A method for fabrication of multiplexed optical coding nanobeads (MOCNBs) was developed by hybridizing three types of coding DNAs labeled with different dyes (Cy5, FAM and AMCA) at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated nanobeads with a diameter of 300 nm. The color of the MOCNBs could be observed by overlapping three single-primary-color fluorescence images of the MOCNBs corresponding to emission of Cy5 (red), FAM (green) and AMCA (blue). The MOCNBs could be easily identified under a conventional fluorescence microscope. The MOCNBs with different colors could serve as the multiplexed optical coding labels for single-molecule counting analysis (SMCA) and be used in multi-gene expression analysis (MGEA). In the SMCA-based MGEA technique, multiple messenger RNAs (mRNAs) in cells could be simultaneously quantified through their complementary DNAs (cDNAs) by counting the bright dots with the same color corresponding to the single cDNA molecules labeled with the MOCNBs. We measured expression profiles of three genes from Lepidoptera insect Helicoverpa armigera in ∼100 HaEpi cells with and without steroid hormone inductions to demonstrate the SMCA-based MGEA technique using MOCNBs.

ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging.

Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research.

Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3×10(-16) mol L(-1). The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of cDNAs corresponding to beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein, large, P2 mRNAs in single human breast cancer cells and 5 random synthetic DNAts are simultaneously quantified to examine the SMA and SMA-based single-cell multiple gene expression analysis.

Gene expression profile of staphylococcal enterotoxin B and lipopolysaccharide induced human PBMCs (799.1)

Staphylococcal enterotoxin B (SEB), an exotoxin produced by staphylococcal aureus, is commonly associated with food poisoning and capable of triggering flu‐like symptoms resembling those of endotoxins such as lipopolysaccharide (LPS) released from Gram‐negative bacterial cell walls. Even though both exhibit analogous symptoms, each has its unique gene expression profile. Here we have successfully distinguished two toxins that display similar symptoms in human peripheral blood mononuclear cells (PBMCs) using multiple gene expression analysis. PBMCs are the most easily accessible biofluid, and include informative transcripts as a first line of immune defense for many disease processes and toxin exposures. Thirty genes that provided a unique genetic signature to differentiate exposures to the two toxins were first identified through microarray analysis and then validated by using multiple gene and protein quantitation techniques. We believe that this study will allow to differentiate two similar toxins and also to better predict various disease states.

Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe

In this study, we introduce the double-barrel carbon probe (DBCP)-a simple, affordable microring electrode-which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from high-volume samples such as spheroids. This method achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.

Development and Characterization of a Clinically Compliant Xeno-Free Culture Medium in Good Manufacturing Practice for Human Multipotent Mesenchymal Stem Cells

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.

Gene Expression from Bronchoscopy Obtained Tumour Samples as a Predictor of Outcome in Advanced Inoperable Lung Cancer

BackgroundSeveral studies have shown the prognostic and predictive potential of molecular markers in combined therapy for lung cancer. Most of them referred, however, to operable early stage NSCLC. The aim of the present study is to correlate the expression of multiple mRNA markers in bronchoscopy obtained cancer specimens with clinical outcome of advanced lung cancer.MethodsBronchoscopy cancer specimens were taken from 123 patients with radiological diagnosis of advanced lung tumor. Out of 123 patients 50 were diagnosed with squamous cell cancer, 17 with adenocarcinoma, 12 with NOS, 32 with SCLC and one with large cell neuroendocrinal cancer. In 11 patients other tumours were diagnosed. The group was heterogeneous with respect to clinical stage, performance of the patients and treatment. Quantitative real time PCR was carried out by ABI 7900 HT machine, with Universal Probe Library (Roche) fluorescent probes. The genes selected for the analysis were ERCC1, EGFR, BRCA1, CSF1, CA9, DUSP6, STAT1, ERBB3, MMD, FN1, and CDKN1B.ResultsMore than 50 ng of RNA (the amount considered sufficient for the analysis) was isolated in 82 out of 112 lung cancer specimens (73%), including 60/80 (75.0%) of NSCLC specimens and 22/32 (68,7%) of SCLC samples. The highest Cohen’s κ coefficient for discrimination between small cell, squamous cell and adenocarcinoma was found for CDKN1B, CSF and EGFR1 (κ = 0.177, p = 0.0041). A multivariate Cox regression model has shown a significant impact of clinical stage (p<0.001, RR = 4.19), ERCC1 (p = 0.01, RR = 0.43) and CA9 (p = 0.03, RR = 2.11) expression on overall survival in a group of 60 patients with NSCLC.ConclusionThese results show the feasibility of multiple gene expression analysis in bronchoscopy obtained cancer specimens as prognostic markers in radiotherapy and chemotherapy for advanced lung cancer. A limiting factor was relatively high proportion of samples from which sufficient amount of RNA could not be isolated.

A novel multiplex polymerase chain reaction assay for profile analyses of gene expression in peripheral blood

BackgroundStudies have demonstrated that inflammation has a key role in the pathogenesis of atherosclerosis due to the abnormal gene expressions of multiple cytokines. We established an accurate and precise method to observe gene expression in whole blood that might provide specific diagnostic information for coronary artery disease (CAD) and other related diseases.MethodsThe fifteen selected CAD-related genes (IL1B, IL6, IL8, IFNG, MCP-1, VWF, MTHFR, SELL, TNFalpha, ubiquitin, MCSF, ICAM1, ID2, HMOX1 and LDLR) and two housekeeping genes (ACTB and GK) as internal references have been measured simultaneously with a newly developed multiplex polymerase chain reaction (multi-PCR) method. Moreover, the precision was evaluated, and a procedure for distinguishing patients from the normal population has been developed based upon analyses of peripheral blood. A total of 148 subjects were divided into group A (control group without plaques), group B (calcified plaques) and group C (non-calcified plaques, and combination group) according dual-source CT criteria. Gene expression in blood was analyzed by multi-PCR, and levels of glucose and lipids measured in 50 subjects to explore the relationship among them.ResultsThe precision results of the multi-PCR system revealed within-run and between-run CV values of 3.695–12.537% and 4.405–13.405%, respectively. The profiles of cytokine gene expression in peripheral blood were set: a positive correlation between glucose and MCSF, HMOX1 or TNFalpha were found. We also found that triglyceride levels were negatively correlated with SELL gene expression in 50 subjects. Compared with controls, gene expression levels of IL1B, IL6, IL8 and MCP-1 increased significantly in group C.ConclusionsA new multiple gene expression analysis system has been developed. The primary data suggested that gene expression was related to CAD. This system might be used for risk assessment of CVDs and other related diseases.