We developed a technology for detecting and quantifying trace nucleic acids using a bracketing protocol designed to yield a copy number with approximately ± 20% accuracy across all concentrations. The microRNAs (miRNAs) let-7b, miR-15b, miR-21, miR-375 and miR-141 were measured in serum and urine samples from healthy subjects and patients with breast, prostate, or pancreatic cancer. Detection and quantification were amplification-free and enabled using osmium-tagged probes and MinION, a nanopore array detection device. Combined serum from healthy men (Sigma‒Aldrich #H6914) was used as a reference. Total RNA isolated from biospecimens using commercial kits was used as the miRNA source. The unprecedented ± 20% accuracy led to the conclusion that miRNA copy numbers must be normalized to the same RNA content, which in turn illustrates (i) independence from age, sex, and ethnicity, as well as (ii) equivalence between serum and urine. miR-21, miR-375 and miR-141 copies in cancers were 1.8-fold overexpressed, exhibited zero overlap with healthy samples and had a p value of 1.6x10-22, tentatively validating each miRNA as a cancer biomarker. miR-15b was confirmed to be cancer-independent, whereas let-7b appeared to be a cancer biomarker for prostate and breast cancer, but not for pancreatic cancer.

Anastassia Kanavarioti is the Founder and Director of Yenos Analytical LLC, a company that delivers custom analytical solutions for native, synthetic, or transcribed nucleic acids and engages in the development and manufacturing of labeled/tagged nucleic acids for use in conjunction with nanopore detection and nucleic acid quantification, including miRNAs, with awarded patents for Nanopore platform for DNA/RNA oligo detection using an osmium tagged complementary probe, Patent No: US 11,111,527 B1, issued 9-7-2021 and US 11,427,859, issued 9-30-2022 and Osmium Tagged Probes for Nucleic Acid Detection, US 11,884,968, issued 01-30-2024. In 2023, international patents were filed in Europe, Canada, Australia, Japan, China, India, Brazil, and South Korea. A provisional patent application entitled Detection, Quantification and Validation of microRNA Biomarkers was filed with USPTO on May 7, 2024. There are no competing interests or relationships (commercial, financial, or nonfinancial) between the two companies, namely Oxford Nanopore Technologies and Yenos Analytical LLC, and their employees.

This study did not receive any funding

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Ethics committee IRB Advarra waived ethical approval for this work. The project to include Urine samples collected from consenting healthy subjects was reviewed by the Advarra Investigational Review Board (IRB). The protocol and consent form were reviewed, modified, and approved by the Advarra IRB on November 15, 2023. Protocol: Yenos Analytical LLC-02. Quantification of selected microRNAs in the urine of healthy individuals (Pro00074065). Donors of urine samples reviewed and signed an informed consent form. They were then sent a kit/insulated box, cold bricks, and instructions to collect their biospecimen at home, freeze it and ship it overnight to the Yenos facilities.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors