The Salivaomics Knowledge Base (SKB) is designed to serve as a computational infrastructure that can permit global exploration and utilization of data and information relevant to salivaomics. SKB is created by aligning (1) the saliva biomarker discovery and validation resources at UCLA with (2) the ontology resources developed by the OBO (Open Biomedical Ontologies) Foundry, including a new Saliva Ontology (SALO).
We define the Saliva Ontology (SALO; http://www.skb.ucla.edu/SALO/) as a consensus-based controlled vocabulary of terms and relations dedicated to the salivaomics domain and to saliva-related diagnostics following the principles of the OBO (Open Biomedical Ontologies) Foundry.
The Saliva Ontology is an ongoing exploratory initiative. The ontology will be used to facilitate salivaomics data retrieval and integration across multiple fields of research together with data analysis and data mining. The ontology will be tested through its ability to serve the annotation ('tagging') of a representative corpus of salivaomics research literature that is to be incorporated into the SKB.
There is a need recognized by the National Institute of Dental & Craniofacial Research and the National Cancer Institute to advance basic, translational and clinical saliva research. The goal of the Salivaomics Knowledge Base (SKB) is to create a data management system and web resource constructed to support human salivaomics research. To maximize the utility of the SKB for retrieval, integration and analysis of data, we have developed the Saliva Ontology and SDxMart. This article reviews the informatics advances in saliva diagnostics made possible by the Saliva Ontology and SDxMart.
BioMart; database; ontology; saliva
Saliva, the most accessible and noninvasive biofluid of our body, harbors a wide spectrum of biological analytes informative for clinical diagnostic applications. While proteomic constituents are a logical first choice as salivary diagnostic analytes, genomic targets have emerged as highly informative and discriminatory. This awareness, coupled with the ability to harness genomic information by high-throughput technology platforms such as genome-wide microarrays, ideally positions salivary genomic targets for exploring the value of saliva for detection of specific disease states and augmenting the diagnostic and discriminatory value of the saliva proteome for clinical applications. Buccal cells and saliva have been used as sources of genomic DNA for a variety of clinical and forensic applications. For discovery of disease targets in saliva, the recent realization that there is a transcriptome in saliva presented an additional target for oral diagnostics. All healthy subjects evaluated have approximately 3,000 different mRNA molecules in their saliva. Almost 200 of these salivary mRNAs are present in all subjects. Exploration of the clinical utility of the salivary transcriptome in oral cancer subjects shows that four salivary mRNAs (OAZ, SAT, IL8, and IL1b) collectively have a discriminatory power of 91% sensitivity and specificity for oral cancer detection. Data are also now in place to validate the presence of unique diagnostic panels of salivary mRNAs in subjects with Sjöogren's disease.
human saliva transcriptome analysis; mRNA biomarkers; noninvasive disease detection
For the past two decades, salivary diagnostic approaches have been developed to monitor oral diseases such as periodontal diseases and to assess caries risk. Recently, the combination of emerging biotechnologies and salivary diagnostics has extended the range of saliva-based diagnostics from the oral cavity to the whole physiological system as most compounds found in blood are also present in saliva. Accordingly saliva can reflect the physiological state of the body, including emotional, endocrinal, nutritional and metabolic variations and provides a source for the monitoring of oral and also systemic health. This review presents the current status of saliva diagnostics and delves into their applications to the discovery of biomarkers for cancer detection and therapeutic applications. Translating scientific findings of nucleic acids, proteins and metabolites in body fluids to clinical applications is a cumbersome and challenging journey. Our research group is pursuing the biology of salivary analytes and the development of technologies in order to detect distinct biomarkers with high sensitivity and specificity. The avenue of saliva diagnostics incorporating transcriptomic, proteomic and metabolomic findings will enable us to connect salivary molecular analytes to monitor therapies, therapeutic outcomes, and finally disease progression in cancer.
saliva diagnostics; biomarker; transcriptome; proteome; therapeutic perspectives
The salivary proteome is a complex protein mixture resulting from the activity of salivary glands with the contribution of other components that form the oral environment such as oral tissues and micro-organisms. For diagnosis purposes, saliva collection has the great advantage of being an easy and non-invasive technique. Human saliva proteomics have proven to be a novel approach in the search for protein biomarkers for detection of different local and systemic diseases. Currently, more than 1400 salivary proteins have been identified. In the last few years, our research group has extensively studied the salivary proteomics in order to analyse the salivary composition, investigating the major families of proteins present in human and mammalian saliva, the post-translational modifications, the different contributions of glands, the physiological and pathological modifications of saliva. The aim of this report is to present our personal experience in salivary proteomics. In conclusion, salivary proteome analysis represents an important field both for diagnosis and monitoring of various diseases and could be considered a novel approach to prevention of various pathological conditions.
Saliva; Salivary glands; Proteomics; Salivary composition; HPLC-ESI-MS
Saliva is a useful biofluid for the early detection of disease, but how distal tumors communicate with the oral cavity and create disease-specific salivary biomarkers remains unclear. Using an in vitro breast cancer model, we demonstrated that breast cancer-derived exosome-like microvesicles are capable of interacting with salivary gland cells, altering the composition of their secreted exosome-like microvesicles. We found that the salivary gland cells secreted exosome-like microvesicles encapsulating both protein and mRNA. We also showed that the interaction with breast cancer-derived exosome-like microvesicles communicated and activated the transcriptional machinery of the salivary gland cells. Thus, the interaction altered the composition of the salivary gland cell-derived exosome-like microvesicles on both the transcriptomically and proteomically.
The proteome of human saliva can be considered as being essentially completed. Diagnostic markers for a number of diseases have been identified among salivary proteins and peptides, taking advantage of saliva as an easy-to-obtain biological fluid. Yet, the majority of disease markers identified so far are serum components and not intrinsic proteins produced by the salivary glands. Furthermore, despite the fact that saliva is essential for protecting the oral integuments and dentition, little progress has been made in finding risk predictors in the salivary proteome for dental caries or periodontal disease. Since salivary proteins, and in particular the attached glycans, play an important role in interactions with the microbial world, the salivary glycoproteome and other post-translational modifications of salivary proteins need to be studied. Risk markers for microbial diseases, including dental caries, are likely to be discovered among the highly glycosylated major protein species in saliva. This review will attempt to raise new ideas and also point to under-researched areas that may hold promise for future applicability in oral diagnostics and prediction of oral disease.
bacterial adhesion; dental; glycomics; oral; proteome; saliva; salivary proteins
Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be non-invasively collected, it provides an attractive alternative
for blood, serum or plasma. It has been postulated that the blood concentrations of many components are reflected in saliva. Saliva harbors a wide array of
proteins, which can be informative for the detection of diseases. Profiling the proteins in saliva over the course of disease progression could reveal
potential biomarkers indicative of different stages of diseases, which may be useful in medical diagnostics. With advanced instrumentation and developed
refined analytical techniques, proteomics is widely envisioned as a useful and powerful approach for salivary proteomic biomarker discovery. As
proteomic technologies continue to mature, salivary proteomics have great potential for biomarker research and clinical applications. The progress and
current status of salivary proteomics and its application in the biomarker discovery of oral and systematic diseases will be reviewed. The scientific and
clinical challenges underlying this approach will also be discussed.
Saliva; Biomarkers; Proteomics
Proteomics studies in saliva have demonstrated its potential as a diagnostic biofluid, however the salivary peptidome is less studied. Here we study the effects of several sample collection and handling factors on salivary peptide abundance levels.
Salivary peptides were isolated using an ultrafiltration device and analyzed by tandem mass spectrometry. A panel of 41 peptides common after various treatments were quantified and normalized. We evaluated the effects of freezing rate of the samples, nutritional status of the donors (fed vs fasted), and room-temperature sample degradation on peptide abundance levels. Repeatability of our sample processing method and our instrumental analysis method were investigated.
Increased sample freezing rate produced higher levels of peptides. Donor nutritional status had no influence on the levels of measured peptides. No significant difference was detected in donors’ saliva following 5, 10 and 15 min of room-temperature degradation. Sample processing and instrumental variability were relatively small, with median CVs of 9.6 and 6.6.
Peptide abundance levels in saliva are rather forgiving towards variations in sample handling and donor nutritional status. Differences in freezing methods may affect peptide abundance, so consistency in freezing samples is preferred. Our results are valuable for standardizing sample collection and handling methods for peptidomic-based biomarker studies in saliva.
Peptidomics; saliva; sample handling; stability; normalization LC-MS/MS
The proteome of human salivary fluid has the potential to open new doors for disease biomarker discovery. A recent study to comprehensively identify and catalog the human ductal salivary proteome led to the compilation of 1166 proteins. The protein complexity of both saliva and plasma is large, suggesting that a comparison of these two proteomes will provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides. To create a more comprehensive catalog of human salivary proteins, we have first compiled an extensive list of proteins from whole saliva (WS) identified through MS experiments. The WS list is thereafter combined with the proteins identified from the ductal parotid, and submandibular and sublingual (parotid/SMSL) salivas. In parallel, a core dataset of the human plasma proteome with 3020 protein identifications was recently released. A total of 1939 nonredundant salivary proteins were compiled from a total of 19 474 unique peptide sequences identified from whole and ductal salivas; 740 out of the total 1939 salivary proteins were identified in both whole and ductal saliva. A total of 597 of the salivary proteins have been observed in plasma. Gene ontology (GO) analysis showed similarities in the distributions of the saliva and plasma proteomes with regard to cellular localization, biological processes, and molecular function, but revealed differences which may be related to the different physiological functions of saliva and plasma. The comprehensive catalog of the salivary proteome and its comparison to the plasma proteome provides insights useful for future study, such as exploration of potential biomarkers for disease diagnostics.
Biomarkers; Body fluid; MS; Plasma; Saliva
Cellular heterogeneity arising from stochastic expression of genes, proteins and metabolites is a fundamental principle of cell biology, but single cell analysis has been beyond the capabilities of ‘Omics’ technologies. This is rapidly changing with the recent examples of single cell genomics, transcriptomics, proteomics and metabolomics. The rate of change is expected to accelerate owing to emerging technologies that range from micro/nanofluidics to microfabricated interfaces for mass spectrometry to third- and fourth-generation automated DNA sequencers. As described in this review, single cell analysis is the new frontier in Omics, and single cell Omics has the potential to transform systems biology through new discoveries derived from cellular heterogeneity.
Recent advancement in mass spectrometry leads us to a new era of proteomic analysis. Human saliva can be easily collected; however, the complexity of the salivary proteome in the past prevented the use of saliva for proteomic analysis. Here we review the development of proteomic analyses for human saliva and focus on the use of a new mass spectrometric technology known as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). SELDI-TOF, a modification of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF), combines the precision of mass spectrometry and the high-through-put nature of protein arrays known as Protein Chips. This technology shows a promising future for salivary proteomic analysis in monitoring treatments and diseases, as well as novel biomarker discovery.
Proteomics; mass spectrometry; biomarker; biomarker discovery; proteomic profiling; protein expression; SELDI; SELDI-TOF; protein chips.
A sensitive assay to identify biomarkers using non-invasively collected clinical specimens is ideal for breast cancer detection. While there are other studies showing disease biomarkers in saliva for breast cancer, our study tests the hypothesis that there are breast cancer discriminatory biomarkers in saliva using de novo discovery and validation approaches. This is the first study of this kind and no other study has engaged a de novo biomarker discovery approach in saliva for breast cancer detection. In this study, a case-control discovery and independent preclinical validations were conducted to evaluate the performance and translational utilities of salivary transcriptomic and proteomic biomarkers for breast cancer detection.
Salivary transcriptomes and proteomes of 10 breast cancer patients and 10 matched controls were profiled using Affymetrix HG-U133-Plus-2.0 Array and two-dimensional difference gel electrophoresis (2D-DIGE), respectively. Preclinical validations were performed to evaluate the discovered biomarkers in an independent sample cohort of 30 breast cancer patients and 63 controls using RT-qPCR (transcriptomic biomarkers) and quantitative protein immunoblot (proteomic biomarkers). Transcriptomic and proteomic profiling revealed significant variations in salivary molecular biomarkers between breast cancer patients and matched controls. Eight mRNA biomarkers and one protein biomarker, which were not affected by the confounding factors, were pre-validated, yielding an accuracy of 92% (83% sensitive, 97% specific) on the preclinical validation sample set.
Our findings support that transcriptomic and proteomic signatures in saliva can serve as biomarkers for the non-invasive detection of breast cancer. The salivary biomarkers possess discriminatory power for the detection of breast cancer, with high specificity and sensitivity, which paves the way for prediction model validation study followed by pivotal clinical validation.
Biochemical systems biology augments more traditional disciplines, such as genomics, biochemistry and molecular biology, by championing (i) mathematical and computational modeling; (ii) the application of traditional engineering practices in the analysis of biochemical systems; and in the past decade increasingly (iii) the use of near-comprehensive data sets derived from ‘omics platform technologies, in particular “downstream” technologies relative to genome sequencing, including transcriptomics, proteomics and metabolomics. The future progress in understanding biological principles will increasingly depend on the development of temporal and spatial analytical techniques that will provide high-resolution data for systems analyses. To date, particularly successful were strategies involving (a) quantitative measurements of cellular components at the mRNA, protein and metabolite levels, as well as in vivo metabolic reaction rates, (b) development of mathematical models that integrate biochemical knowledge with the information generated by high-throughput experiments, and (c) applications to microbial organisms. The inevitable role bioinformatics plays in modern systems biology puts mathematical and computational sciences as an equal partner to analytical and experimental biology. Furthermore, mathematical and computational models are expected to become increasingly prevalent representations of our knowledge about specific biochemical systems.
Saliva (oral fluids) is an emerging biofluid poised for detection of clinical diseases. Although the rationale for oral diseases applications (e.g. oral cancer) is intuitive, the rationale and relationship between systemic diseases and saliva biomarkers are unclear.
In this study, we used mouse models of melanoma and non-small cell lung cancer and compared the transcriptome biomarker profiles of tumor-bearing mice to those of control mice. Microarray analysis showed that salivary transcriptomes were significantly altered in tumor-bearing mice vs. controls. Significant overlapping among transcriptomes of mouse tumors, serum, salivary glands and saliva suggests that salivary biomarkers have multiple origins. Furthermore, we identified that the expression of two groups of significantly altered transcription factors (TFs) Runx1, Mlxipl, Trim30 and Egr1, Tbx1, Nr1d1 in salivary gland tissue of melanoma-bearing mice can potentially be responsible for 82.6% of the up-regulated gene expression and 62.5% of the down-regulated gene expression, respectively, in the saliva of melanoma-bearing mice. We also showed that the ectopic production of nerve growth factor (NGF) in the melanoma tumor tissue as a tumor-released mediator can induce expression of the TF Egr-1 in the salivary gland.
Taken together, our data support the conclusion that upon systemic disease development, significant changes can occur in the salivary biomarker profile. Although the origins of the disease-induced salivary biomarkers may be both systemic and local, stimulation of salivary gland by mediators released from remote tumors plays an important role in regulating the salivary surrogate biomarker profiles.
The capability to assess physiological states, detect morbidity initiation and progression, and monitor post-treatment therapeutic outcomes through a noninvasive approach is one of the most desirable goals for healthcare research and delivery. Saliva, a multi-constituent oral fluid, has high potential for the surveillance of general health and disease. To reach the above goal through saliva-based diagnostics, two prerequisites must be fulfilled: (1) discovering biomarker(s) for different diseases among the complicated components of saliva, and (2) advancing sensitivity and specificity of biomarker(s) through persistent development of technologies. Under the support and research blueprint initiated by the National Institute of Dental and Craniofacial Research (NIDCR), salivary diagnostics has not only steadily progressed with respect to accuracy and availability, but has also bridged up-to-date nanotechnology to expand the areas of application. With collective efforts over several years, saliva has been demonstrated to be a promising bodily fluid for early detection of diseases, and salivary diagnostics has exhibited tremendous potential in clinical applications. This review presents an overview of the value of saliva as a credible diagnostic tool, the discovery of salivary biomarkers, and the development of salivary diagnostics now and in the future.
Since the advent of the new proteomics era more than a decade ago, large-scale studies of protein profiling have been used to identify distinctive molecular signatures in a wide array of biological systems, spanning areas of basic biological research, clinical diagnostics, and biomarker discovery directed toward therapeutic applications. Recent advances in protein separation and identification techniques have significantly improved proteomic approaches, leading to enhancement of the depth and breadth of proteome coverage.
Proteomic signatures, specific for multiple diseases, including cancer and pre-invasive lesions, are emerging. This article combines, in a simple manner, relevant proteomic and OMICS clues used in the discovery and development of diagnostic and prognostic biomarkers that are applicable to all clinical fields, thus helping to improve applications of clinical proteomic strategies for translational medicine research.
Saliva plays a major role in maintaining oral health. Patients afflicted with a decrease in saliva secretion (symptomatically, xerostomia) exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. Despite recent improvements in treating xerostomia (e.g., saliva stimulants, saliva substitutes, and gene therapy), there is a need of more scientific advancements that can be clinically applied toward restoration of compromised salivary gland function. Here we provide a summary of the current salivary cell models that have been used to advance restorative treatments via development of an artificial salivary gland. These models represent initial steps toward clinical and translational research, to facilitate creation of clinically safe salivary glands. Further studies in salivary cell lines and primary cells are necessary to improve survival rates, cell differentiation, and secretory function. Additionally, the characterization of salivary progenitor and stem cell markers are necessary. Although these models are not fully characterized, their improvement may lead to the construction of an artificial salivary gland that is in high demand for improving the quality of life of many patients suffering from salivary secretory dysfunction.
cell line; progenitor cells; primary culture; salivary gland dysfunction
There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins—histatin 5, statherin, and PRP1—was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.
biochemistry; biomarkers; enzymology; proteolysis; saliva; salivary diagnostics
The use of human saliva as a diagnostic and prognostic fluid has until recently been somewhat disregarded. Although sample collection is non-invasive, physiological and genetic variations were largely responsible for its infrequent application in the past. Recently, several proteomic studies contributed to partial elucidation of the salivary proteome (more than 2400 protein components have been characterized), both in terms of composition, contributions to whole saliva and genetic/physiological variability. On this basis, is not too optimistic to believe that in the near future human saliva could become a relevant diagnostic fluid. In this review, the characterization by proteomic approaches of new salivary markers in oncology, head and neck carcinoma (oral cavity, oropharynx, larynx, and salivary glands), breast and gastric cancers, salivary gland function and disease, Sjögren syndrome, systemic sclerosis, dental and gingival pathology, systemic, psychiatric and neurological diseases, is described.
Saliva; Salivary glands; Proteins; Peptides; Proteomics; Oral cavity; Oncology; Dental pathology; Systemic diseases; Therapy
Salivary diagnostics has fascinated many researcheres and has been tested as a valuable tool in the diagnosis of many systemic conditions and for drug monitoring. Advances in the field of molecular biology, salivary genomics and proteomics have led to the discovery of new molecular markers for oral cancer diagnosis, therapeutics and prognosis. Oral cancer is a potentially fatal disease and the outcome of the treatment and prognosis largely depends on early diagnosis. Abnormal cellular products elucidated from malignant cells can be detected and measured in various body fluids including saliva and constitute tumor markers. This article discusses the various salivary tumor markers and their role in oral pre-cancer and cancer.
DNA markers; oral cancer; protein markers; RNA markers; salivary markers; tumor markers
Recent advancements in mass spectrometric proteomics provide a promising result in utilizing saliva to explore biomarkers for diagnostic purposes. However, the issues of specificity or redundancy of disease-associated salivary biomarkers have not been described. This systematic review was therefore aimed to define and summarize disease-related salivary biomarkers identified by mass spectrometry proteomics. Peer-reviewed articles published through July 2009 within three databases were reviewed. Out of 243 articles, 21 studies were selected in this systematic review with conditions including Sjögren's syndrome, squamous cell carcinoma, dental caries, diabetes, breast cancer, periodontitis, gastric cancer, systemic sclerosis, oral lichen planus, bleeding oral cavity, and graft-versus-host disease. The sample size ranged from 3–41 in both diseased and control subjects, with no consensus on sample collection protocol. One hundred eighty biomarkers were identified in total; 87 upregulated, 63 downregulated, and 30 varying based on disease. Except for Sjögren's syndrome, the majority of studies with the same disease produce inconsistent biomarkers. Larger sample size and standardization of sample collection/treatment protocol may improve future studies.
Over the last two decades, advanced molecular genetics technology has enabled analysis of complex microbial communities and the study of microbial genomics. Interest has grown in characterizing the microbiome, defined as a collective microbial community and its extensive genome, as a clue to disease mechanisms. “The Human Microbiome Project,” sponsored by the NIH Common Fund, was established to characterize the pathology-associated human microbiome in nasal passages, oral cavities, skin, the gastrointestinal tract, and the urogenital compartment. In particular, characterization of urogenital microbiota may elucidate etiologies of complex obstetrical syndromes and factors in fetal development that define risk for pathology in adulthood. This article summarizes recent findings defining the microbiome associated with the female urogenital compartment in child-bearing age women. We also describe our analysis of microbiome samples from the oral, vaginal, and rectal compartments in a cohort of pregnant women. Findings present technical considerations in the characterization of microbial diversity and composition associated with gestational diabetes as a model pregnancy-associated pathology.
Micro-organisms; microbiology; microbiota; microbiome; metagenome; gynecology; obstetrics
In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.
Submandibular/Sublingual saliva; MudPIT; lectin-affinity chromatography; glycoproteins; biomarkers
Salivary biomarkers for diagnostic and prognostic assessments have become increasingly well established in recent years. Salivary mRNA transcriptomic analyses create a new paradigm in the emerging field for noninvasive molecular diagnosis. In this chapter, we will overview the development of sensitive and robust microarray and multiplex quantitative reverse transcriptase-PCR assays for the discovery and validation of mRNA biomarkers in human saliva. Total RNA isolated from human saliva is used for microarray profiling through Human Genome U133 Plus 2.0 and Exon 1.0 ST array platforms. A universal RNA linear amplification strategy was used to amplify RNA from nanogram scale followed by reverse transcription-PCR reaction, cleaned up enzymatically, and validated by quantitative PCR. Further, the integrity of RNA can be analyzed by the Agilent Bioanalyzer and quantified using a Nanodrop microvolume spectrophotometer. Using these invaluable technical tools, one can identify thousands of mRNA species in saliva. These methods indicate that salivary mRNA provides an efficient medium for biomarker discovery in oral and systemic diseases detection.
Saliva; mRNA profiling; biomarkers; oral cancer; microarray profile; mRNA stability