RT-qPCR for Fecal Mature MicroRNA Quantification and Validation.

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase (RT) followed by TaqMan® minor-groove binding (MGB) probe, real-time quantitative PCR analysis on exfoliated enriched colonocytes in stool, using human (Homo sapiens, hsa) micro(mi)RNAs to monitor changes of their expression at various stages of colorectal (CRC) progression, this method allows for the reliable and quantitative diagnostic screening of colon cancer (CC). Although the expression of some miRNA genes tested in tissue shows less variability in normal or cancerous patients than in stool, the noninvasive stool by itself is well suited for CC screening. An miRNA approach using stool promises to offer more sensitivity and specificity than currently used genomic, methylomic, or proteomic methods for CC screening.To present an application of employing miRNAs as diagnostic markers for CC screening, we carried out global microarray expression studies on stool colonocytes isolated by paramagnetic beads, using Affymetrix GeneChip miRNA 3.0 Array, to select a panel of miRNAs for subsequent focused semiquantitative PCR analysis studies. We then conducted a stem-loop RT-TaqMan® MGB probes, followed by a modified real-time qPCR expression study on 20 selected miRNAs for subsequent validation of the extracted immunocaptured total small RNA isolated from stool colonocytes. Results showed 12 miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p, and miR214) to have an increased expression in stool of CC patients, and that later TNM stages exhibited more increased expressions than adenomas, while 8 miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222, and miR-938) showed decreased expressions in stool of CC patients, which becomes more pronounced as the cancer progresses from early to late TNM stages (0-IV).

Role of Melt Curve Analysis in Interpretation of Nutrigenomics' MicroRNA Expression Data.

This article illustrates the importance of melt curve analysis (MCA) in interpretation of mild nutrogenomic micro(mi)RNA expression data, by measuring the magnitude of the expression of key miRNA molecules in stool of healthy human adults as molecular markers, following the intake of Pomegranate juice (PGJ), functional fermented sobya (FS), rich in potential probiotic lactobacilli, or their combination. Total small RNA was isolated from stool of 25 volunteers before and following a three-week dietary intervention trial. Expression of 88 miRNA genes was evaluated using Qiagen's 96 well plate RT2 miRNA qPCR arrays. Employing parallel coordinates plots, there was no observed significant separation for the gene expression (Cq) values, using Roche 480® PCR LightCycler instrument used in this study, and none of the miRNAs showed significant statistical expression after controlling for the false discovery rate. On the other hand, melting temperature profiles produced during PCR amplification run, found seven significant genes (miR-184, miR-203, miR-373, miR-124, miR-96, miR-373 and miR-301a), which separated candidate miRNAs that could function as novel molecular markers of relevance to oxidative stress and immunoglobulin function, for the intake of polyphenol (PP)-rich, functional fermented foods rich in lactobacilli (FS), or their combination. We elaborate on these data, and present a detailed review on use of melt curves for analyzing nutigenomic miRNA expression data, which initially appear to show no significant expressions, but are actually more subtle than this simplistic view, necessitating the understanding of the role of MCA for a comprehensive understanding of what the collective expression and MCA data collectively imply.

miRNA as markers for the diagnostic screening of colon cancer.

Early screening for colon cancer (CC) allows for early stage diagnosis of the malignancy and potentially reduces disease mortality as the cancer is most likely curable at its earliest stages. Early detection would be desirable if accurate, practical and cost-effective diagnostic measures for this cancer were available. Mortality and morbidity from CC represent a major health problem involving a malignant disease that is theoretically preventable through screening. Current screening methods (e.g., the convenient and inexpensive immunological fecal occult blood test, FOBTi, obtained from patients' medical records) either lack sensitivity and require dietary restriction, which impedes compliance and use; are costly (e.g., colonoscopy), which decreases compliance; or could result in mortality. In comparison with the FOBT test, a non-invasive sensitive screen for which there is no requirement for dietary restriction would be a more convenient test. Colorectal cancer is the only cancer for which colonoscopy is recommended as a screening method. Although colonoscopy is a reliable screening tool, the invasive nature, abdominal pain, potential complications and high cost have hampered the application of this procedure worldwide. A screening approach using the stable miRNA molecules, which are relatively non-degradable when extracted from non-invasive stool and semi-invasive blood samples by commercially available kits and manipulated thereafter, would be preferable to a transcriptomic mRNA-, a mutation DNA-, an epigenetic- or a proteomic-based test. The approach uses reverse transcriptase, modified real-time quantitative PCR. Although exosomal RNA would be missed, using a restricted extraction of total RNA from stool or blood, a parallel test could also be carried out on RNA obtained from stool or plasma samples, and appropriate corrections for exsosomal loss can be made for accurate and quantitative test result. Eventually, a chip can be developed to facilitate diagnosis, as has been done for the quantification of genetically modified organisms in foods. The gold standard to which the molecular miRNA test is compared is colonoscopy, which can be obtained from patients' medical records. If performance criteria are met, as detailed herein, a miRNA test in human stool or blood samples based on high-throughput automated technologies and quantitative expression measurements commonly used in the diagnostic clinical laboratory should be advanced to the clinical setting, which will make a significant impact on CC prevention.

Diagnostic microRNA markers to screen for sporadic human colon cancer in stool: I. Proof of principle.

To present proof-of-principle application for employing micro(mi)RNAs as diagnostic markers for colon cancer, we carried out global microarray expression studies on stool samples obtained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer), using Affymetrix GeneChip miRNA 3.0 Array, to select for a panel of miRNA genes for subsequent focused semi-quantitative polymerase chain reaction (PCR) analysis studies. Microarray results showed 202 preferentially expressed miRNA genes that were either increased (141 miRNAs), or reduced (61 miRNAs) in expression. We then conducted a stem-loop reverse transcriptase (RT)-TaqMan® minor groove binding (MGB) probes, followed by a modified qPCR expression study on 20 selected miRNAs. Twelve of the miRNAs exhibited increased and 8 decreased expression in stool from 60 individuals (20 controls, 20 with tumor-lymph node-metastatic (TNM) stage 0-1, 10 with stage 2, five with stage 3, and 5 with stage 4 colon cancer) to quantitatively monitor miRNA changes at various TNM stages of colon cancer progression. We also used laser-capture microdissection (LCM) of colon mucosal epithelial tissue samples (three control samples, and three samples from each of the four stages of colon cancer, for a total of 15 samples) to find concordance or lack thereof with stool findings. The reference housekeeping pseudogene-free ribosomal gene (18S rRNA), which shows little variation in expression, was employed as a normalization standard for relative PCR quantification. Results of the PCR analyses confirmed that twelve miRNAs (miR-7, miR-17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p and miR214) had an increased expression in the stool of patients with colon cancer, and that later TNM carcinoma stages exhibited a more pronounced expression than did adenomas. On the other hand, eight miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222 and miR-938) had decreased expression in the stool of patients with colon cancer, which was also more pronounced from early to later TNM stages. Results from colon mucosal tissues were similar to those from stool samples, although with more apparent changes in expression. Cytological studies on purified stool colonocytes that employed Giemsa staining showed 80% sensitivity for detecting tumor cells in stool smears. The performance characteristics of the test confirmed that stool is a medium well-suited for colon cancer screening, and that the quantitative changes in the expression of few mature miRNA molecules in stool associated with colon cancer progression provided for more sensitive and specific non-invasive diagnostic markers than tests currently available on the market. Thus, a larger prospective and properly randomized validation study of control individuals and patients exhibiting various stages of colon cancer progression (TNM stages 0-IV) is now needed in order to standardize test conditions, and provide a means for determining the true sensitivity and specificity of a miRNA screening approach in stool for the non-invasive detection of colon cancer, particularly at an early stage (0-I). Eventually, we will develop a chip to enhance molecular screening for colon cancer, as has been accomplished for the detection of genetically-modified organisms (GMOs) in foods.

Diagnostic microRNA markers to screen for sporadic human colon cancer in blood.

We carried out this study to present proof-of-principal application, showing that by using a global microarray expression analysis, followed by quantitative stem-loop reverse transcriptase in conjunction with TaqMan® polymerase chain reaction (PCR) analysis of micro(mi)RNA genes, on limited number of plasma and tissue samples obtained from 20 individuals (five healthy, five TNM stage 0-1 colon cancer, five stage 2 and five stage 3), we were able to quantitatively monitor miRNA changes at the various TNM stages of colon cancer progression, particularly at the early, pre-malignant adenoma stage (e.g. polyps ≥ 1 cm with high grade dysplasia). The expression of some of the tested miRNAs showed less variability in tissue than in plasma. Nevertheless, our limited preliminary data on the plasma by itself show that plasma is well-suited for screening, and that the quantitative changes in the expression of a few cell-free circulatory mature miRNA molecules in plasma, that are associated with colon cancer progression, would provide for more sensitive and specific markers than those tests currently available on the market. In addition, analysis of miRNA molecules offers a quantitative and cost-effective non-invasive diagnostic approach for screening, than currently employed methods in a prevalent cancer that can be cured if it is detected at the early TNM stages, and that becomes deadly if not diagnosed before metastasis. Thus, a larger prospective and properly randomized clinical study using plasma derived from many control individuals and at various stages of colon cancer (TNM stages 0-IV) from patients, in order to corroborate the initial results, is now urgently needed in order to allow for a statistically valid analysis, standardizing test conditions which will provide a means for determining the true sensitivity and specificity of a miRNA-screening approach. This approach, when combined with bioinformatics analysis to correlate miRNA seed data with mRNA target data, would allow for a mechanistic understanding of how miRNAs regulate mRNA gene expression, and would offer a better comprehensive diagnostic screening test for early-detection of colon cancer non-invasively.

Surface plasmon resonance (SPR) spectrometry as a tool to analyze nucleic acid-protein interactions in crude cellular extracts.

This study presents proof-of-principle application showing that label-free affinity enrichment surface plasmon resonance (SPR) biosensor binding is able to semiquantitatively detect molecular DNA-protein interactions in crude cellular extracts in a real-time ligand fishing analysis study. Crude cell extracts obtained from a confluent HT-28 human adenocarcinoma cell line, synchronized to the G(0)/G(1) phase of the cell cycle, were extracted in a chaotropic medium and cryopreserved in liquid nitrogen. Various immunoprecipitation antibodies were used against defective human excision and mismatch repair genes, hDDB2 and hMSH2, respectively, which theoretically allow for protein binding to DNA ligands in their native conformation. A set of biotinylated DNA target sequence heteroduplexes were also utilized for binding hDDB2 and hMSH2, prepared by heating a biotinylated oligonucleotide strand with an equimolar amount of the complementary strand to form a DNA duplex for hMSH2; a UV-irradiated duplex was employed for hDDB2 instead of an irradiated single-strand DNA to enhance binding. SDS was used to regenerate heteroduplex-modified chips that were used in a BIAcore 2000 SPR instrument at 25°C. Results showed that hMSH2 does not bind preferentially to the heteroduplex-complementary pair. In contrast, hDDB2 was found to bind preferentially to the UV-irradiated version of the heteroduplex-complementary pair. It is concluded that the choice of antibodies with appropriate epitopes is crucial to the success of these SPR binding studies because of enhanced specificity.

Diagnostic microRNA markers for screening sporadic human colon cancer and active ulcerative colitis in stool and tissue.

By routinely and systematically being able to perform quantitative stem-loop reverse transcriptase followed by TaqMan PCR expression analysis on stool and tissue samples using fifteen human (Homo sapiens, hsa) micro(mi)RNA genes selected by careful analysis of the peer-reviewed literature, we were able to monitor changes at various stages of CRC, allowing for reliable diagnostic screening of colon cancer particularly at the early, pre-malignant stages, and for difficult-to-treat active ulcerative colitis (UC). Although the expression of some of the miRNA genes tested in tissue showed less variability in CRC or UC patients than in stool, the stool by itself appears well-suited to screening. A miRNA approach using stool samples promises to offer more sensitivity and specificity than currently used screening genomic, methylomic or proteomic methods for colon cancer. Larger prospective clinical studies utilizing stool derived from many control, colon cancer or UC patients, to allow for a statistically valid analysis, are now urgently required to standardize test performance and determine the true sensitivity and specificity of the miRNA screening approach, and to provide a numerical underpinning for these diseases as a function of total RNA. Moreover, when a miRNA screening test is combined with analysis of a messenger(m)RNA expression test, which has also been considered in earlier studies to be a highly sensitive and a very specific and reliable transcriptomic approach, as outlined in this article, bioinformatics can be used to correlate microRNA seed data with mRNA target data in order to gain a mechanistic understanding of how miRNAs regulate gene expression, enabling understanding of why these miRNA genes should be informative in a screening test.

The role of capillary electrophoresis-mass spectrometry to proteome analysis and biomarker discovery.

CE offers a low running cost, short separation time, and a high-resolution technique that requires only a small amount of analyte. It has a wide variety of operation modes (CZE, CEC, CIEF, CITP, CAE, CGE and MEKC) that can be interfaced with MS for tissue and body fluid analysis, particularly urine and cerebrospinal fluid, to identify potential proteomic markers for the clinical diagnosis of many diseases (renal, genitourinary, vascular, diabetes mellitus, cancer, arthritis and neurological diseases) and for the monitoring of their therapeutic intervention. It has become evident that no one marker would be sufficient, but a combination of well-selected markers would be needed for that purpose. The potential of CE coupled to MS for studying the pathophysiology of these diseases and the development of biomarkers has been demonstrated. These biomarkers, when validated, will allow greater use of noninvasive methods for diagnosis of diseases, assessment of their progression, and monitoring individuals' response to therapy.

Differences in mRNA and microRNA microarray expression profiles in human colon adenocarcinoma HT-29 cells treated with either Intensity-modulated Radiation Therapy (IMRT), or Conventional Radiation Therapy (RT).

We carried out this in vitro molecular study to investigate the effect of two clinical X-irradiation modalities (a two-dimensional external beam radiotherapy referred to in this article as conventional RT, and a three dimensional conformal intensity-modulated radiation therapy (IMRT) on a colon adenocarcinoma HT-29 cell line. Cells were synchronized by serum deprivation 48 h before irradiation so that >90% of them were in the G(0)/G(1) phase of the cell cycle. Cells were allowed to recover 3 h after irradiation before total RNA extraction. Two types of arrays, namely Affymetrix Human HG U133A 2.0 oligonucleotide microarrays and Ambion mirVana bioarrays, were employed to study mRNA and microRNA expressions, respectively. Three flasks were used per irradiation dose, and an additional three unirradiated flasks served as control. Microarray data were validated by reverse transcriptase quantitative polymerase chain reaction, and proteins of some expressed genes were determined by Western blots. Results showed the existence of differences in expression profiles between the two irradiation modalities. IMRT appeared to influence expression of some DNA repair genes, whereas in conventional RT, some DNA repair and cell cycle-related genes that initially seemed to be preferentially expressed dwindled to normal levels. Earlier in vitro experiments using cell survival to study sublethal damage repair support our conclusions. Bioinformatic investigation revealed a correlation of gene expression with derepression effects of microRNA molecules. We have presented opinions as to how microRNAs might influence gene expression during radiation-induced stress and have suggested future avenues for research.

Utility of mass spectrometry for proteome analysis: part II. Ion-activation methods, statistics, bioinformatics and annotation.

This is the second article in a series, intended as a tutorial to provide the interested reader with an overview of the concepts not covered in part I, such as: the principles of ion-activation methods, the ability of mass-spectrometric methods to interface with various proteomic strategies, analysis techniques, bioinformatics and data interpretation and annotation. Although these are different topics, it is important that a reader has a basic and collective understanding of all of them for an overall appreciation of how to carry out and analyze a proteomic experiment. Different ion-activation methods for MS/MS, such as collision-induced dissociation (including postsource decay) and surface-induced dissociation, electron capture and electron-transfer dissociation, infrared multiphoton and blackbody infrared radiative dissociation have been discussed since they are used in proteomic research. The high dimensionality of data generated from proteomic studies requires an understanding of the underlying analytical procedures used to obtain these data, as well as the development of improved bioinformatics tools and data-mining approaches for efficient and accurate statistical analyses of biological samples from healthy and diseased individuals, in addition to determining the utility of the interpreted data. Currently available strategies for the analysis of the proteome by mass spectrometry, such as those employed for the analysis of substantially purified proteins and complex peptide mixtures, as well as hypothesis-driven strategies, have been elaborated upon. Processing steps prior to the analysis of mass spectrometry data, statistics and the several informatics steps currently used for the analysis of shotgun proteomic experiments, as well as proteomics ontology, are also discussed.

Sample preparation and fractionation for proteome analysis and cancer biomarker discovery by mass spectrometry.

Sample preparation and fractionation technologies are one of the most crucial processes in proteomic analysis and biomarker discovery in solubilized samples. Chromatographic or electrophoretic proteomic technologies are also available for separation of cellular protein components. There are, however, considerable limitations in currently available proteomic technologies as none of them allows for the analysis of the entire proteome in a simple step because of the large number of peptides, and because of the wide concentration dynamic range of the proteome in clinical blood samples. The results of any undertaken experiment depend on the condition of the starting material. Therefore, proper experimental design and pertinent sample preparation is essential to obtain meaningful results, particularly in comparative clinical proteomics in which one is looking for minor differences between experimental (diseased) and control (nondiseased) samples. This review discusses problems associated with general and specialized strategies of sample preparation and fractionation, dealing with samples that are solution or suspension, in a frozen tissue state, or formalin-preserved tissue archival samples, and illustrates how sample processing might influence detection with mass spectrometric techniques. Strategies that dramatically improve the potential for cancer biomarker discovery in minimally invasive, blood-collected human samples are also presented.

Liquid chromatography-mass spectrometry: a tool for proteome analysis and biomarker discovery and validation.

BACKGROUND: Mass spectrometry (MS) coupled to liquid chromatography (LC) represents a widely used analytical tool for studying proteins, and discovering various diseases biomarkers. OBJECTIVES/METHODS: To enable interested readers to grasp fundamental and experimental concepts of liquid chromatography separation for protein analytes and their accurate detection when interfaced with the powerful technique of mass spectrometry. Methods for enhancing LC separation, the utility of an atmospheric pressure soft ionization method, particularly electrospray ionization, as well as the interphase of improved chromatographic columns with the various modes of mass spectrometry, and the characteristics of the various mass analyzers, will be elucidated in order to achieve sensitive and specific detection of protein markers. CONCLUSIONS: With a coherent biomarker pipeline, the successful throughput from a discovery phase to a robust validation method has nevertheless been disappointing. The development of faster, cost-effective and sensitive multidimensional MS methods, improvements in sample preparation and processing procedures such as high abundance protein depletion and faster chromatographic columns and modes, in addition to optimization of MS-based detection methods, are expected to improve further the analytical performance of proteomic techniques for biomarker discovery in complicated, but convenient, non- or minimally invasive clinical samples such as blood and urine, and ultimately to improve the biomarker validation process.

Utility of mass spectrometry for proteome analysis: part I. Conceptual and experimental approaches.

This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.

Mining the oncoproteome and studying molecular interactions for biomarker development by 2DE, ChIP and SPR technologies.

This article provides a comprehensive updated review of three proteomics technologies (2D gel electrophoresis [2DE]; chromatin immunoprecipitation [ChIP] and its related alternates, and surface plasmon resonance [SPR]), and their use in cancer biomarker discovery and studying molecular interactions. 2DE proteomics has an advantage in visualizing changes in the intact molecular weight and isoelectric point (pI) of a protein, which reflect biologically significant processing and charge alterations in addition to post-translation modifications. However, proteins that are hydrophobic, low abundance or with extreme pIs or molecular weight, are poorly represented. Despite these shortfalls, improvements have been made that provide enhanced resolution by use of immobilized pH gradients (IPGs), narrow strip large 2DE gels, and serial and parallel formats. Prefractionation of the sample is essential for studying low-abundance markers, although it subjects the experimental design to additional nonbiologic variations. 2DE has been applied to the successful identification of oncoproteins in serum and tissues of cancers, such as colon, breast, prostate and pancreas. Interactions between protein and DNA are essential for cellular function. The process of developing global approaches to studying chromatin began with the in vitro characterization of chromatin structural components by the versatile ChIP assay and its modifications, which are capable of analyzing protein-DNA interactions in vivo. These methods have been used to assess gene expression, transcription factor binding and/or histone modifications in various organisms, including human. Label-free, real-time analysis of interactions in biological systems employing SPR biosensors that measure changes in refractive index of solvents near the surface during complex formation or dissociation of proteins, oligonucleotides, oligosaccharides and lipids to small molecules, phage, viral particles and cells is used extensively in research laboratories and the pharmaceutical and biotechnology industries for studying drug discovery, biopharmaceutical developments and clinical immunogenicity assays, as well as in manufacturing and quality-control methods.

Role of miRNA in carcinogenesis and biomarker selection: a methodological view.

miRNAs, their involvement in cancer development and their potential to be robust biomarkers of diagnosis, staging, prognosis and response to therapy are reviewed. In small RNA animal biogenesis, miRNA genes in the nucleus are transcribed to generate long primary transcripts (pri-miRNAs), which are first cropped by RNase-III-type enzyme Drosha to release hairpin intermediates (pre-miRNAs) in the nucleus. Pre-miRNA is then exported to the cytoplasm by exportin-5. Following arrival in the cytoplasm, pre-miRNAs are subjected to the second processing step (dicing) to release the mature miRNA duplex, which is then separated: one strand becomes the mature miRNA and the other is degraded. These tiny miRNAs induce messenger degradation, translational repression or both. However, there is no evidence to demonstrate that these two mechanisms exist in the regulation of the same gene. Since a miRNA can target numerous mRNAs, often in combination with other miRNAs, these miRNAs operate a highly complex regulatory network. The specific function in most mammalian miRNAs is unknown. However, data suggest that miRNA genes, approximately 1% of all human genes, regulate protein production for 20-30% or more of all genes. miRNA expression profiles are effective for classifying solid and hematologic human cancers, and have shown great promise for early cancer detection. This is of great importance for effective treatment before the cells metastasize; therefore, tumors can be surgically resected. Computer-based prediction approaches of miRNAs and their targets, and biological validation techniques for ascertaining these predictions, currently play a central role in the discovery of miRNAs and in elucidating their function. Guidelines have been established for the identification and annotation of new miRNAs to distinguish them from other RNAs, especially siRNAs. These guidelines take into account factors such as transcript structure, conservation and processing, and a centralized, searchable database of all possible miRNA sequence information and annotation for humans and of more than 38 other species. Two approaches are used to characterize miRNAs: studying expression of known miRNAs by hybridization-based techniques (e.g., northern blots, RNase protection, primer extension, real-time, quantitative PCR and microarrays) or discovery of novel miRNAs molecules by cloning and sequencing. Owing to their adaptability and high throughput, microarrays may prove to be the preferred platform for whole-genome miRNA expression analysis.

Transcriptomic molecular markers for screening human colon cancer in stool and tissue.

There is a need for sensitive and specific diagnostic molecular markers that can be used to monitor early patterns of gene expression in non-invasive exfoliated colonocytes shed in the stool, and in situ in adenoma-carcinoma epithelium of the colon. RNA-based detection methods are more comprehensive than either DNA-, protein- or methylation-based screening methods. By routinely and systematically being able to perform quantitative gene expression studies on these samples using less than ten colon cancer genes selected by the enormous resources of the National Cancer Institute's Cancer Genome Anatomy Project, we were able to monitor changes at various stages in the neoplastic process, allowing for reliable diagnostic screening of colon cancer particularly at the early, pre-malignant stages. Although the expression of some of the genes tested in tissue showed less variability in normal or cancerous patients than in stool, the stool by itself is suitable for screening. Thus, a transcriptomic approach using stool or tissue samples promises to offer more sensitivity and specificity than currently used molecular screening methods for colon cancer. A larger prospective clinical study utilizing stool and tissue samples derived from many control and colon cancer patients, to allow for a statistically valid analysis, is now urgently required to determine the true sensitivity and specificity of the transcriptomic screening approach for this preventable cancer.

Colorectal cancer epigenetics: the role of environmental factors and the search for molecular biomarkers.

This review presents an evenhanded evaluation of the role of epigenetics in the development of colorectal cancer, and investigates the extent of environmental influences on modulating this disease. Advances in our understanding of chromatin structure, histone modification, transcriptional activity and DNA methylation have lead to an integrated approach to the role of epigenetics in carcinogenesis. Epigenetic mechanisms appear to permit response of individuals to environment through change in gene expression and are involved in inactivating one of the two X chromosomes in women. Epigenetic changes play an important role in development and can also arise stochastically as individuals age. Because epigenetic alterations are potentially reversible, thereby allowing malignant cells to revert to the normal state, there is potential to develop effective strategies to prevent or even reverse this curable cancer. Moreover, because the methylation status of a specific sequence or the pattern of methylation across the genome can now be measured accurately, molecular biomarkers of screening, diagnosis, prognosis, prediction of treatment and those related to risk assessment can be developed using sophisticated molecular genetic technologies. Although in many cases a high sensitivity and specificity of the detection assays has been achieved, there still remains ample room for improvement in areas of sample preparation, assay design and marker selection.

Modeling survival in colon cancer: a methodological review.

The Cox proportional hazards model is the most widely used model for survival analysis because of its simplicity. The fundamental assumption in this model is the proportionality of the hazard function. When this condition is not met, other modifications or other models must be used for analysis of survival data. We illustrate in this review several methodological approaches to deal with the violation of the proportionality assumption, using survival in colon cancer as an illustrative example.

Standardization for transcriptomic molecular markers to screen human colon cancer.

Establishing test performance criteria for a transcriptomic colon cancer marker approach must be carried out in a standardized fashion in order tso ensure that the test will perform the same way in any laboratory, anywhere. Condition of sample preservation and shipping prior to total RNA extraction is critical, and we recommend preserving stool samples in an appropriate preservative and shipping them in cold packs so as to keep stools at 4 degrees C. It is not necessary to isolate colonocytes to obtain adequate RNA for testing. It is, however, important to obtain samples from both mucin-rich and non-mucin rich to have a good representation of both left- and right-side colon cancers. Employing a commercial total RNA extraction kit that contains an RLT buffer from Qiagen Corporation (Valencia, CA, USA) removes bacterial RNA from stool preparations and results in a high yield of undegraded RNA of human origin. Genes selected based on the enormous resources of NCI's Cancer Genome Anatomy project give good results. Primers for PCR should span more than one exon. Use of semiquantitative PCR, preferably with several reference housekeeping genes of various copy numbers, depending on tested genes, should enhance confidence in the quantitative results. Having standardized the testing conditions in our ongoing work, it is now imperative that a larger prospective randomized clinical study utilizing stool and tissue samples derived from several control and colon cancer patients, to allow for statistically valid analyses, be conducted in order to determine the true sensitivity and specificity of the transcriptomic screening approach for this cancer whose incidence is on the rise worldwide.

Microarray RNA transcriptional profiling: part II. Analytical considerations and annotation.

This review summarizes the various data filtration, transformation and normalization processes for different array platforms (cDNA, oligos, one- and two-color), data analysis methods and their validation, and databases and annotation for RNA transcriptional profiling microarrays. This review is intended to introduce the beginner to the analyses and interpretation of gene expression studies using a nonmathematical approach for easier comprehension. Microarray analysis is not a trivial undertaking as there is no single method that works well for all, and results obtained from these analyses should be considered as a complement to other approaches.