Methods for Sequencing Cell-Free RNA

{
  "publication_number": "US2026103749A1",
  "country": "US",
  "kind": "A1",
  "title": "Methods for Sequencing Cell-Free RNA",
  "abstract": "In an aspect, a method comprises obtaining cell-free ribonucleic acid (RNA) from a plasma sample of a subject; converting the cell-free RNA into complementary deoxyribonucleic acid (cDNA), thereby producing sample cDNA; and sequencing the sample eDNA to determine a level of the sample cDNA that corresponds to a set of RNA transcripts.",
  "claims": [
    "1. A method comprising: (a) obtaining a whole blood sample from a subject; (b) fractionating the whole blood sample, wherein the fractionating comprises centrifuging the whole blood sample to obtain a plasma sample of the subject; (c) extracting cell-free ribonucleic acid (RNA) from the plasma sample of the subject; (d) converting the cell-free RNA into complementary deoxyribonucleic acid (cDNA) by performing reverse transcription, thereby producing sample cDNA; (e) amplifying the sample cDNA to produce amplified DNA; (f) sequencing the amplified DNA to produce sequence information; (g) quantitating the sequence information to determine a level of the sample cDNA that corresponds to a set of RNA transcripts comprising brain-derived RNA transcripts, wherein the determining comprises selecting for a subset of the sequence information corresponding to the set of RNA transcripts, wherein the set of RNA transcripts is differentially expressed in plasma samples of a first population of subjects with Alzheimer's disease as compared to plasma samples of a second population of subjects without Alzheimer's disease; (h) computer processing the level of the sample cDNA determined in (g) (1) against a reference level or (2) using a trained classification model; and (i) determining that the subject has the Alzheimer's disease, based at least in part on the computer processing in (h). 2. The method of claim 1, wherein (c) further comprises extracting total RNA from the plasma sample. 3. The method of claim 2, wherein extracting the total RNA comprises contacting the plasma sample with chloroform to produce a mixture. 4. The method of claim 3, further comprising centrifuging the mixture to produce an aqueous layer. 5. The method of claim 4, further comprising isolating the total RNA from the aqueous layer. 6. The method of claim 5, further comprising performing DNase digestion of the total RNA. 7 - 8. (canceled) 9. The method of claim 1, wherein the whole blood sample has a volume of 7 milliliters to 15 milliliters. 10 - 12. (canceled) 13. The method of claim 1, wherein the amplification comprises polymerase chain reaction (PCR). 14. The method of claim 13, wherein the PCR comprises indiscriminate PCR. 15. The method of claim 1, wherein the sequencing comprises whole transcriptome sequencing. 16. The method of claim 1, further comprising performing microarray analysis of the sample cDNA. 17. The method of claim 1, further comprising performing quantitative polymerase chain reaction (PCR) analysis of the sample cDNA. 18. The method of claim 1, wherein the reference level comprises a level of cDNA corresponding to a population of subjects without Alzheimer's disease. 19. The method of claim 1, wherein the reference level comprises a level of cDNA corresponding to a population of subjects diagnosed with Alzheimer's disease. 20. The method of claim 1, wherein the reference level corresponds to a level of cDNA corresponding to one or more housekeeping genes. 21. The method of claim 20, wherein the one or more housekeeping genes comprise actin beta (ACTB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 22. The method of claim 1, wherein the reference level corresponds to one or more up-regulated genes. 23. The method of claim 22, wherein the one or more up-regulated genes comprise amyloid beta precursor protein (APP) or pleckstrin and sec7 domain containing 3 (PSD3). 24. The method of claim 1, wherein the reference level corresponds to one or more down-regulated genes. 25. The method of claim 24, wherein the one or more down-regulated genes comprise myelin-associated oligodendrocyte basic protein (MOBP), myelin associated glycoprotein (MAG), solute carrier family 2 member 1 (SLC2A1), transcription factor 7-like 2 (TCF7L2), cadherin 22 (CDH22), ciliary neurotrophic factor (CNTF), or progestin and adipoQ receptor family member 6 (PAQR6). 26. The method of claim 1, wherein (h) further comprises computer processing the level of the sample cDNA determined in (g) using the trained classification model. 27. The method of claim 1, wherein (h) further comprises comparing the level of the sample cDNA determined in (g) (1) against the reference level or (2) using the trained classification model. 28. The method of claim 1, wherein the determining in (i) comprises determining a combined z-score based on a statistical difference between the level of the sample cDNA determined in (g) and the reference level."
  ],
  "description_excerpt": "The present invention relates to assessing neurological disorders based on nucleic acid specific to brain tissue.\n\nDementia is a catchall term used to characterize cognitive declines that interfere with one's ability to perform everyday activities. Signs of dementia include declines in the following mental functions: memory, communication and language, ability to focus and pay attention, reasoning, judgment, motor skills, and visual perception. While there are several neurological disorders that cause dementia, Alzheimer's disease is the most common, accounting for 60 to 80 percent of all dementia cases.\n\nAlzheimer's disease is a progressive disease that gradually destroys memory and mental functions in patients. Symptoms manifest initially as a decline in memory followed by deterioration of other cognitive functions as well as by abnormal behavior. Individuals with Alzheimer's disease usually begin to show dementia symptoms later in life (e.g., 65 years or older), but a small percentage of individuals in their 40s and 50s experience early onset Alzheimer's disease. Alzheimer's disease is associated with the damage and degeneration of neurons in several regions of the brain. The neuropathic characteristics of Alzheimer's disease include the presence of plaques and tangles, synaptic loss, and selective neuronal cell death. Plaques are abnormal levels of protein fragments called beta-amyloid that accumulate between nerve cells. Tangles are twisted fibers of a protein known as tau that accumulate within nerve cells.",
  "cpc": [
    "C12Q 1/686",
    "C12Q 1/6806"
  ],
  "ipc": [
    "C12Q 1/6806",
    "C12Q 1/686",
    "C12Q 1/68"
  ],
  "assignees": [
    "Leland Stanford Junior University"
  ],
  "inventors": [
    "Lian Chye Winston Koh",
    "Stephen R. Quake",
    "Hei-Mun Christina Fan",
    "Wenying Pan"
  ],
  "filing_date": "2024-10-11",
  "publication_date": "2026-04-16",
  "priority_date": "2012-01-27",
  "application_number": "US-202418913930-A",
  "family_id": "72145550",
  "cited_by_count": 0,
  "citations": [
    "US20070179197A1",
    "WO2012145363A1"
  ]
}