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Transcriptomic analyses of saliva.

ABSTRACT: 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.

SUBMITTER: Palanisamy V

PROVIDER: S-EPMC3171800 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Transcriptomic analyses of saliva.

Palanisamy Viswanathan V Wong David T DT

Methods in molecular biology (Clifton, N.J.) 20100101

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 f ...[more]

PMID: 20717777

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Project description:The detection of SARS-CoV-2 biomarkers by real time PCR (rRT-PCR) has shown that the sensitivity of the test is negatively affected by low viral loads and the severity of the disease. This limitation can be overcome by the use of more sensitive approaches such as mass spectrometry (MS), which has not been explored for the detection of SARS-CoV-2 proteins in saliva. Thus, this study aimed at assessing the translational applicability of mass spectrometry-based proteomics approaches to identify viral proteins in saliva from people diagnosed with COVID-19 within fourteen days after the initial diagnosis, and to compare its performance with rRT-PCR. After ethics approval, saliva samples were self-collected by 42 COVID-19 positive and 16 healthy individuals. Samples from people positive for COVID-19 were collected on average on the sixth day (± 4 days) after initial diagnosis. Viable viral particles in saliva were heat-inactivated followed by the extraction of total proteins and viral RNA. Proteins were digested and then subjected to tandem MS analysis (LC-QTOF-MS/MS) using a data-dependent MS/MS acquisition qualitative shotgun proteomics approach. The acquired spectra were queried against a combined SARS-CoV-2 and human database. The qualitative detection of SARS-CoV-2 specific RNA was done by rRT-PCR. SARS-CoV-2 proteins were identified in all COVID-19 samples (100%), while viral RNA was detected in only 24 out of 42 COVID-19 samples (57.1%). Seven out of 18 SARS-CoV-2 proteins were identified in saliva from COVID-19 positive individuals, from which the most frequent were replicase polyproteins 1ab (100%) and 1a (91.3%), and nucleocapsid (45.2%). Neither viral proteins nor RNA were detected in healthy individuals. Our mass spectrometry approach appears to be more sensitive than rRT-PCR for the detection of SARS-CoV-2 biomarkers in saliva collected from COVID-19 positive individuals up to 14 days after the initial diagnostic test. Based on the novel data presented here, our MS technology can be used as an effective diagnostic test of COVID-19 for initial diagnosis or follow-up of symptomatic cases, especially in patients with reduced viral load.

Dataset Information

Transcriptomic analyses of saliva.

Publications

Transcriptomic analyses of saliva.

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