Methods for profiling and quantitating cell-free RNA
Inventors
Koh, Lian Chye Winston • Quake, Stephen R. • Fan, Hei-Mun Christina • Pan, Wenying
Assignees
Leland Stanford Junior University
Publication Number
US-12365947-B2
Publication Date
2025-07-22
Expiration Date
2033-01-28
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Abstract
The invention generally relates to methods for assessing the health of a tissue by characterizing circulating nucleic acids in a biological sample. According to certain embodiments, methods for assessing the health of a tissue include the steps of detecting a sample level of RNA in a biological sample, comparing the sample level of RNA to a reference level of RNA specific to the tissue, determining whether a difference exists between the sample level and the reference level, and characterizing the tissue as abnormal if a difference is detected.
Core Innovation
The invention describes methods for assessing the health of tissues or organs by characterizing circulating nucleic acids, specifically cell-free RNA, present in biological samples such as blood plasma. It solves the problem of requiring invasive biopsies or expensive imaging techniques to monitor organ health by providing a non-invasive method to detect tissue-specific RNA transcripts released into the bloodstream, which reflect tissue health status.
Methods include detecting levels of tissue-specific RNA in a biological sample and comparing them to reference levels derived from healthy individuals or specific patient populations. Deviations in these relative RNA contributions indicate abnormal tissue health, such as organ failure, immune attack, or pathological conditions. The invention also enables comprehensive profiling of fetal-specific cell-free RNA in maternal plasma to monitor fetal development and pregnancy complications using high throughput sequencing and microarray technologies.
Claims Coverage
The claims include one independent claim focused on a multi-step method for detecting and analyzing cell-free mRNA in blood plasma, incorporating preparative, amplification, sequencing, and analytical steps.
Preparation and amplification of enriched cell-free mRNA from blood plasma
Obtaining a blood plasma sample with cell-membrane stabilizing agents to deplete cells, preparing complementary DNA (cDNA) from cell-free messenger RNA (mRNA) in the blood plasma, and amplifying the prepared cDNA indiscriminately to produce double-stranded cDNA without dilution or sample partitioning.
Sequencing and quantifying cell-free mRNA from specific genes
Sequencing the double-stranded cDNA to generate sequence information and determining levels of cell-free mRNA associated with a specific group of genes (ABHD8, CHST10, CLCN6, FBXL15, GPR17, KIF5A, KLHL26, NELL1, PIAS4, PPP2R2D, RAB33A, RAC3, RAPGEFL1, THAP10, and TIMM22), representing whole brain tissue origin.
Comparing expression levels and characterizing abnormalities
Determining the contribution of the cell-free mRNA levels relative to an expression level of a reference gene expressed in blood plasma, and characterizing the gene as abnormal if a difference is detected between the sample and the reference levels.
Sample processing by centrifugation
Conducting sequential centrifugation steps, with a first centrifugation at or above 1600×g and a second centrifugation at or above 16,000×g to separate cells and residual cells from blood sample to yield plasma for analysis.
The claims cover a method for non-invasive detection and quantitation of tissue-specific cell-free mRNA in plasma by combined cell stabilization, RNA isolation, amplification, sequencing, and comparative analysis to reference gene expression levels to assess tissue health or abnormalities.
Stated Advantages
Allows non-invasive assessment of tissue health by analyzing circulating RNA without relying on invasive biopsies or disease-related protein biomarkers.
Enables simultaneous screening of multiple tissue-specific transcriptomes from limited biological samples with single-nucleotide resolution.
Facilitates early detection of pathological conditions, including cancer, pregnancy complications, developmental diseases, and organ transplant rejection, by quantifying deviations in tissue-specific RNA contributions.
Provides a method to characterize fetal development and identify pregnancy-associated transcripts non-invasively across gestational timepoints.
Documented Applications
Non-invasive diagnosis and monitoring of cancer screening, tumor initiation and progression through detecting circulating tumor-derived RNA markers in blood.
Prenatal molecular diagnostics by comprehensive profiling of fetal-specific cell-free RNA in maternal plasma across trimesters to monitor fetal development and detect pregnancy complications such as premature birth or preeclampsia.
Screening and monitoring for organ health and abnormalities including appendicitis, diabetes-related pathologies, graft versus host disease in bone marrow or solid organ transplants, and neurological disorders involving cell-specific death or protein aggregation.
Quantitative assessment and prognostic monitoring of fetal tissue apoptosis rates and developmental diseases involving abnormal fetal organ growth or cell death.
Use in diagnostics involving multiple biological fluids and tissues, including blood, stool, sputum, urine, transvaginal fluid, breast nipple aspirate, cerebrospinal fluid, and tissue biopsy samples.
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