A Comparison of miRNA Isolation and RT-qPCR Technologies and their Effects on Quantification Accuracy and Repeatability

Redshaw, Nicholas; Wilkes, Timothy; Whale, Alexandra S.; Cowen, Simon; Huggett, Jim F.; Foy, Carole A.

doi:10.2144/000114002

Cited by 113 publications

(92 citation statements)

References 47 publications

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“…MiRNA quantification by both methods, however, showed difference in variability that impact miRNA measurements, and therefore quantification is influenced by the choice of assay methodology. Thus, the method used for quantification must be considered when interpreting analyses of PCR results [77][78][79][80].…”

Section: Current Methods For Colon Cancer Screeningmentioning

confidence: 99%

MicroRNAs as molecular markers for colon cancer Diagnostic screening in stool & blood

Ahmed¹,

Ahmed²

2017

Med Res Innov

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Screening for colon cancer (CC) allows for diagnosis of the early stage for malignancy and potentially reduces disease mortality as the cancer could be cured at disease earliest stages. Early detection could be desirable if accurate, practical and cost effective diagnostic measures for this cancer are available. Mortality and morbidity from colon cancer represent a major health problem involving a malignant disease that is theoretically preventable through screening. Current screening methods (e.g., the immunological fecal occult blood test, FOBTi, obtained from patients' medical records) either lacks sensitivity and requires dietary restriction, which impedes compliance and use; are costly (e.g., colonoscopy), which decreases compliance; or could lead to mortality. In comparison to the FOBT test, a noninvasive sensitive screen for which there is no requirement for dietary restriction would be a more convenient test. Colorectal cancer (CRC) is the only cancer for which screening by colonoscopy is recommended. Although colonoscopy is a reliable screening tool, its invasive nature, accompanying abdominal pain, potential complications and high cost have hampered the application of this screening method worldwide.A novel screening approach using the stable miRNA molecules, which are relatively nondegradable when extracted from noninvasive stool and semi-invasive blood samples by currently available commercial kits and manipulated thereafter, would be preferable to a transcriptomic messenger (m)RNA-, a mutation DNA-, an epigenetic-or a proteomic-based test. The approach utilizes reverse transcriptase (RT), followed by a modified quantitative real-time polymerase chain reaction (qPCR). Although exosomal RNA would not be measured, using a restricted extraction of total RNA from stool or blood, then 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 to obtain accurate and quantitative results. Eventually, a chip would be developed to facilitate diagnosis, as has been carried out for the quantification of genetically modified organisms (GMOs) in foods. The gold standard to which the molecular miRNA test is compared is colonoscopy. If performance criteria are met, as detailed herein, then 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--would eventually be advanced to the clinical setting, which will make a vivid impact on the prevention of colon cancer.Correspondence to: Farid E. Ahmed, GEM

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Section: Current Methods For Colon Cancer Screeningmentioning

confidence: 99%

MicroRNAs as molecular markers for colon cancer Diagnostic screening in stool & blood

Ahmed¹,

Ahmed²

2017

Med Res Innov

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“…For 67 commonly detectable miRNAs in plasma and serum, a low correlation between the two qRT-PCR methods was found. A second study revealed that the two methods yielded significantly different copy number estimations of some miRNAs, even though the efficiency appears to be similar [73]. To the best of our knowledge, at present, no consensus has been reached regarding which assay should be preferentially used.…”

Section: Current Limits To the Use Of Circulating Mirnas In Clinical mentioning

confidence: 97%

Circulating microRNAs and diabetes: potential applications in medical practice

et al. 2015

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The explosive increase in the worldwide prevalence of diabetes over recent years has transformed the disease into a major public health concern. While diabetes can be screened for and diagnosed by reliable biological tests based on blood glucose levels, by and large there are no means of detecting at-risk patients or of following diabetic complications. The recent discovery that microRNAs are not only chief intracellular players in many biological processes, including insulin secretion and action, but are also circulating, has put them in the limelight as possible biological markers. Here we discuss the potential role of circulating microRNAs as biomarkers in the context of diabetes and its associated complications.

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“…Other assay (the miRCury LNA Universal RT PCR) involves the poly(A)tailing of mature miRNAs and the subsequent use of a poly (T) reverse transcription primer containing a 3' degenerate anchor and a 5' universal tag to generate cDNA for amplification by SYBR Green qPCR using miRNA-specific forward and reverse primers containing locked nucleic acids (LNAs). The primers are commercially available or easy to design on the basis of the sequence identified by the microarray (46).…”

Section: Biogenesismentioning

confidence: 99%

MicroRNA function in domestic animal physiology and diseases: a promising diagnostic tool for veterinary use

Chodkowska¹,

Sadkowski²,

Ostaszewski³

2017

Medycyna Weterynaryjna

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SummaryMicroRNAs (miRNAs) are small non-coding interfering RNA molecules capable of post-transcriptionally regulating gene expression through sequence-specific base pairing to messenger ribonucleic acid (mRNA). In recent years, hundreds of miRNAs have been identified in humans, various animals and plants. The action of miRNAs has been examined in several biological processes, including tissue morphogenesis, development, cell proliferation and differentiation, apoptosis, immunity, metabolism, and major signaling pathways. Changes in miRNA expression have also been analyzed in the context of various pathological conditions, including different kinds of inflammation, cancer, cardiovascular diseases, etc. Thanks to these investigations, several miRNAs have been identified as potential sensitive diagnostic markers that may be important in monitoring physiological and pathological processes. In human medicine, microarray and real-time PCR-based diagnostic test panels with selected disease-specific miRNAs are increasingly used to predict disease occurrence or progression. In animals, they are mainly used to diagnose canine mammary cancers and infectious diseases, as well as to monitor reproduction. Until now, only few miRNAs of domestic animals have been studied in detail. Moreover, the silencing of selected miRNAs, successfully used in human medicine in diseases related to miRNA over-expression, is also emerging as a promising tool for veterinary medicine and animal breeding.This review presents recent progress in miRNA biology in various domestic animals and shows the current state of knowledge concerning miRNAs and their potential role as a diagnostic factor in veterinary sciences.

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A Comparison of miRNA Isolation and RT-qPCR Technologies and their Effects on Quantification Accuracy and Repeatability

Cited by 113 publications

References 47 publications

MicroRNAs as molecular markers for colon cancer Diagnostic screening in stool & blood

MicroRNAs as molecular markers for colon cancer Diagnostic screening in stool & blood

Circulating microRNAs and diabetes: potential applications in medical practice

MicroRNA function in domestic animal physiology and diseases: a promising diagnostic tool for veterinary use

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