MRNA-based disease diagnosis

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Workflow of blood-based mRNA test on specific cancers. Workflow of Blood-based mRNA Tests on Cancers.tif
Workflow of blood-based mRNA test on specific cancers.

mRNA-based disease diagnosis technologies are diagnostic procedures using messenger RNAs. [1] as molecular diagnostic tools to discover the relationships between patient's DNAs and their specific biological features. The mRNA-based disease diagnosis technologies have been applied to medical field widely in recent years, especially on early diagnosis of tumors (such as renal cell carcinoma, [2] hepatocellular carcinoma, [3] [4] breast cancer [5] and prostate cancer [6] ). The technology can be applied to various types of samples depending on how easily the samples are accessible and whether the samples reliably contain the mRNA that related to specific diseases. For example, in hepatocellular carcinoma, [3] the tumor tissues excised during the operation are a good resource for mRNA-based test to analysis. Among those most commonly used samples , blood sample is one of the most easily accessible via minimally invasive method. degenerative diseases . Blood has been used in early diagnosis of some cancers, [7] [8] such as non-small lung cancer [9] and neuroendocrine tumors. [10]

Contents

Background

Even though modern medicine has been developing for centuries, we are still faced with quite number of medical challenges. For example, in breast cancer, three traditional expressions are routinely screened for target therapy. [11] However, for the triple negative breast cancer, none of those biomarkers can be detected leaving it with poor prognosis and high mortality. Innovative technologies like mRNA are aimed to addressing such health-related issues that still exist today. [12]

Commonly used methods in mRNA-based disease diagnosis

RNA sequencing (RNA-seq)
RNA-seq is a high-throughput RNA sequencing technology that allows scientists to profile the entire RNA (transcriptome). Therefore, novel transcripts and gene expression level can be identified based on cDNA libraries. This method can be used for cancer diagnosis and treatment evaluation. [13]
Reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR is a widely used mRNA expression detection method. It enables reverse transcription of mRNA to cDNA for further identification and qualification. In early 1992, RT-PCR was applied in PSA gene expression in peripheral blood for early prostate cancer diagnosis. [6]
Digital PCR (dPCR)
dPCR is a relatively accurate quantification method of measuring the initial concentration of mRNA targets. It can be applied to analyze genetic and epigenetic changes. [14]
In-situ hybridization (ISH)
ISH is a tissue dependent visualization method of identifying mRNA targeted in the samples. The "tissue" can be blood sample. In chronic myeloid leukemia, ISH has been applied on peripheral-blood specimens. [15]

Workflow

A general workflow of mRNA-based disease diagnosis can be summarized as the following steps: [16] [17] [18]

  1. Sample collection for targeted disease: Samples for mRNA-based technology can be blood, urine, cell culture, tissue biopsy, bronchial alveola lavage, saliva, cerebrospinal fluid. The basic principle to acquire the biological samples for mRNA-based disease diagnosis is that the samples are suitable, accessible, preservable, ethical and minimum invasive. Moreover for some specific research, the samples should be collected within certain time to guarantee the quality and reduce potential contaminants.
  2. RNA processing: This step includes RNA extraction, reverse transcription to cDNA, amplification and detection. The aim to process RNA is to qualify and quantify the suspected genes for further analysis.
  3. Data analysis: After obtaining the qualified raw data from last step, bioinformatics technology will be applied to further analysis the data. There are different tools and software for data analysis, including data normalization, statistical analysis, and modeling. The aim is to select specific genes that are significantly different expressed between disease and non-disease samples. Those selected genes can be identified as diagnostic biomarkers for further validation.
  4. mRNA biomarker validation: The mRNA biomarkers are selected by bioinformatic methods. Thus, they need to be tested with real samples with reliable experiment on real identified samples with clear and accurate clinical diagnosis.
  5. Clinical application: The validated mRNA-based diagnostic method for specific diseases can be conducted along with other clinical examinations to achieve a comprehensive evaluation of the patient's status. mRNA-based examination can provide the medical workers with interpretation at RNA levels and assist on further therapy advice.

Characteristics

High sensitivity

Take breast cancer as an example. The sensitivity of traditional ultrasound screening for breast cancer can be 76%. [19] While with the blood-based mRNA diagnostic method, the sensitivity could be 80.6%. [20]

Qualified and quantitive measurement

mRNA-based disease diagnostic technologies allow quantitive measurement of mRNA in the certain samples, such as leukemia [21]

Guiding personalized therapy

As some technologies such as RNA-seq can provide the entire transcriptome of individual, the mRNA-based disease diagnosis can be developed in the landscape of personalized medicine. In HER-2 breast cancer, detection of ERBB2 mRNA expression levels is helpful in predicting response to anti-HER2-based treatments. [22]

Early diagnostic and predictive method

As mentioned above, the mRNA-based disease diagnostic technology is more sensitive and specific to certain diseases. Even though there is no obvious symptom, the mRNA-based disease diagnostic technology can serve as screening method for early changes in RNA levels. [23] High serum metadherin mRNA expression was observed in colorectal cancer and associated with poorly differentiated histological grades [24]

Related Research Articles

<span class="mw-page-title-main">Reverse transcription polymerase chain reaction</span> Laboratory technique to multiply an RNA sample for study

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

<span class="mw-page-title-main">Keratin 19</span> Protein-coding gene in the species Homo sapiens

Keratin, type I cytoskeletal 19 also known as cytokeratin-19 (CK-19) or keratin-19 (K19) is a 40 kDa protein that in humans is encoded by the KRT19 gene. Keratin 19 is a type I keratin.

In medicine, a biomarker is a measurable indicator of the severity or presence of some disease state. It may be defined as a "cellular, biochemical or molecular alteration in cells, tissues or fluids that can be measured and evaluated to indicate normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention." More generally a biomarker is anything that can be used as an indicator of a particular disease state or some other physiological state of an organism. According to the WHO, the indicator may be chemical, physical, or biological in nature - and the measurement may be functional, physiological, biochemical, cellular, or molecular.

<span class="mw-page-title-main">Keratin 8</span>

Keratin, type II cytoskeletal 8 also known as cytokeratin-8 (CK-8) or keratin-8 (K8) is a keratin protein that is encoded in humans by the KRT8 gene. It is often paired with keratin 18.

Digital polymerase chain reaction is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and qPCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences — such as copy number variants and point mutations.

mir-92 microRNA precursor family

The miR-92 microRNAs are short single stranded non-protein coding RNA fragments initially discovered incorporated into an RNP complex with a proposed role of processing RNA molecules and further RNP assembly. Mir-92 has been mapped to the human genome as part of a larger cluster at chromosome 13q31.3, where it is 22 nucleotides in length but exists in the genome as part of a longer precursor sequence. There is an exact replica of the mir-92 precursor on the X chromosome. MicroRNAs are endogenous triggers of the RNAi pathway which involves several ribonucleic proteins (RNPs) dedicated to repressing mRNA molecules via translation inhibition and/or induction of mRNA cleavage. miRNAs are themselves matured from their long RNA precursors by ribonucleic proteins as part of a 2 step biogenesis mechanism involving RNA polymerase 2.

<span class="mw-page-title-main">Circulating tumor cell</span> Cell from a primary tumor carried by blood circulation

A circulating tumor cell (CTC) is a cell that has shed into the vasculature or lymphatics from a primary tumor and is carried around the body in the blood circulation. CTCs can extravasate and become seeds for the subsequent growth of additional tumors (metastases) in distant organs, a mechanism that is responsible for the vast majority of cancer-related deaths. The detection and analysis of CTCs can assist early patient prognoses and determine appropriate tailored treatments. Currently, there is one FDA-approved method for CTC detection, CellSearch, which is used to diagnose breast, colorectal and prostate cancer.

mIRN21 Non-coding RNA in the species Homo sapiens

microRNA 21 also known as hsa-mir-21 or miRNA21 is a mammalian microRNA that is encoded by the MIR21 gene.

A gene signature or gene expression signature is a single or combined group of genes in a cell with a uniquely characteristic pattern of gene expression that occurs as a result of an altered or unaltered biological process or pathogenic medical condition. This is not to be confused with the concept of gene expression profiling. Activating pathways in a regular physiological process or a physiological response to a stimulus results in a cascade of signal transduction and interactions that elicit altered levels of gene expression, which is classified as the gene signature of that physiological process or response. The clinical applications of gene signatures breakdown into prognostic, diagnostic and predictive signatures. The phenotypes that may theoretically be defined by a gene expression signature range from those that predict the survival or prognosis of an individual with a disease, those that are used to differentiate between different subtypes of a disease, to those that predict activation of a particular pathway. Ideally, gene signatures can be used to select a group of patients for whom a particular treatment will be effective.

Geniom RT Analyzer is an instrument used in molecular biology for diagnostic testing. The Geniom RT Analyzer utilizes the dynamic nature of tissue microRNA levels as a biomarker for disease progression. The Geniom analyzer incorporates microfluidic and biochip microarray technology in order to quantify microRNAs via a Microfluidic Primer Extension Assay (MPEA) technique.

MicroRNA sequencing (miRNA-seq), a type of RNA-Seq, is the use of next-generation sequencing or massively parallel high-throughput DNA sequencing to sequence microRNAs, also called miRNAs. miRNA-seq differs from other forms of RNA-seq in that input material is often enriched for small RNAs. miRNA-seq allows researchers to examine tissue-specific expression patterns, disease associations, and isoforms of miRNAs, and to discover previously uncharacterized miRNAs. Evidence that dysregulated miRNAs play a role in diseases such as cancer has positioned miRNA-seq to potentially become an important tool in the future for diagnostics and prognostics as costs continue to decrease. Like other miRNA profiling technologies, miRNA-Seq has both advantages and disadvantages.

<span class="mw-page-title-main">Cancer biomarker</span> Substance or process that is indicative of the presence of cancer in the body

A cancer biomarker refers to a substance or process that is indicative of the presence of cancer in the body. A biomarker may be a molecule secreted by a tumor or a specific response of the body to the presence of cancer. Genetic, epigenetic, proteomic, glycomic, and imaging biomarkers can be used for cancer diagnosis, prognosis, and epidemiology. Ideally, such biomarkers can be assayed in non-invasively collected biofluids like blood or serum.

mir-618 microRNA is a short non-coding RNA molecule belonging both to the family of microRNAs and to that of small interfering RNAs (siRNAs). MicroRNAs function to regulate the expression levels of other genes by several mechanisms, whilst siRNAs are involved primarily with the RNA interference (RNAi) pathway.

Extracellular RNA (exRNA) describes RNA species present outside of the cells in which they were transcribed. Carried within extracellular vesicles, lipoproteins, and protein complexes, exRNAs are protected from ubiquitous RNA-degrading enzymes. exRNAs may be found in the environment or, in multicellular organisms, within the tissues or biological fluids such as venous blood, saliva, breast milk, urine, semen, menstrual blood, and vaginal fluid. Although their biological function is not fully understood, exRNAs have been proposed to play a role in a variety of biological processes including syntrophy, intercellular communication, and cell regulation. The United States National Institutes of Health (NIH) published in 2012 a set of Requests for Applications (RFAs) for investigating extracellular RNA biology. Funded by the NIH Common Fund, the resulting program was collectively known as the Extracellular RNA Communication Consortium (ERCC). The ERCC was renewed for a second phase in 2019.

<span class="mw-page-title-main">Molecular diagnostics</span> Collection of techniques used to analyze biological markers in the genome and proteome

Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. In medicine the technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients, and in agricultural biosecurity similarly to monitor crop- and livestock disease, estimate risk, and decide what quarantine measures must be taken.

A liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. Like traditional biopsy, this type of technique is mainly used as a diagnostic and monitoring tool for diseases such as cancer, with the added benefit of being largely non-invasive. Liquid biopsies may also be used to validate the efficiency of a cancer treatment drug by taking multiple samples in the span of a few weeks. The technology may also prove beneficial for patients after treatment to monitor relapse.

Circulating free DNA (cfDNA) (also known as cell-free DNA) are degraded DNA fragments released to body fluids such as blood plasma, urine, cerebrospinal fluid, etc. Typical sizes of cfDNA fragments reflect chromatosome particles (~165bp), as well as multiples of nucleosomes, which protect DNA from digestion by apoptotic nucleases. The term cfDNA can be used to describe various forms of DNA freely circulating in body fluids, including circulating tumor DNA (ctDNA), cell-free mitochondrial DNA (ccf mtDNA), cell-free fetal DNA (cffDNA) and donor-derived cell-free DNA (dd-cfDNA). Elevated levels of cfDNA are observed in cancer, especially in advanced disease. There is evidence that cfDNA becomes increasingly frequent in circulation with the onset of age. cfDNA has been shown to be a useful biomarker for a multitude of ailments other than cancer and fetal medicine. This includes but is not limited to trauma, sepsis, aseptic inflammation, myocardial infarction, stroke, transplantation, diabetes, and sickle cell disease. cfDNA is mostly a double-stranded extracellular molecule of DNA, consisting of small fragments (50 to 200 bp) and larger fragments (21 kb) and has been recognized as an accurate marker for the diagnosis of prostate cancer and breast cancer.

Urinary cell-free DNA (ucfDNA) refers to DNA fragments in urine released by urogenital and non-urogenital cells. Shed cells on urogenital tract release high- or low-molecular-weight DNA fragments via apoptosis and necrosis, while circulating cell-free DNA (cfDNA) that passes through glomerular pores contributes to low-molecular-weight DNA. Most of the ucfDNA is low-molecular-weight DNA in the size of 150-250 base pairs. The detection of ucfDNA composition allows the quantification of cfDNA, circulating tumour DNA, and cell-free fetal DNA components. Many commercial kits and devices have been developed for ucfDNA isolation, quantification, and quality assessment.

Smell as evidence of disease has been long used, dating back to Hippocrates around 400 years BCE. It is still employed with a focus on volatile organic compounds (VOCs) found in body odor. VOCs are carbon-based molecular groups having a low molecular weight, secreted during cells' metabolic processes. Their profiles may be altered by diseases such as cancer, metabolic disorders, genetic disorders, infections, and among others. Abnormal changes in VOC composition can be identified through equipment such as gas chromatography-mass spectrometry(GC-MS), electronic nose (e-noses), and trained non-human olfaction.

<span class="mw-page-title-main">MicroRNA biosensors</span>

MicroRNA (miRNA) biosensors are analytical devices that involve interactions between the target miRNA strands and recognition element on a detection platform to produce signals that can be measured to indicate levels or the presence of the target miRNA. Research into miRNA biosensors shows shorter readout times, increased sensitivity and specificity of miRNA detection and lower fabrication costs than conventional miRNA detection methods.

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