Methods to determine the distribution profiles of circulating rnas

A grading method and fractional technology, which can be applied in the fields of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., and can solve problems such as unrevealed miRNA markers.

Inactive Publication Date: 2017-08-18
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, despite extensive screening, such patter

Method used

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  • Methods to determine the distribution profiles of circulating rnas
  • Methods to determine the distribution profiles of circulating rnas
  • Methods to determine the distribution profiles of circulating rnas

Examples

Experimental program
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Embodiment 1

[0084] Chemicals and biological materials. HDL and low density lipoprotein (LDL) were purchased from CalBioChem (EMD Millipore, Billerica, MA). Trizol LS reagent, 3,3'-Dioctadecyloxacarbonylcyanine periodate (DiO) and total exosome isolation kit were purchased from Invitrogen (Life Technologies). MicroRNA standards were purchased from Integrated DNA Technologies (Coralville, IA). TaqMan MicroRNA Assays specific for each miRNA strand were purchased from Applied Biosystems (Life Technologies). For the preparation of 1X PBS (10mM Phosphate, 137mM NaCl, 2.7mM KCl, and 1.0mM MgCl at pH 7.4 2 ), ethylene glycol, dimethyl sulfoxide, guanidine hydrochloride, RNA grade glycogen, 2-propanol, and chloroform were purchased from Thermo Fisher (Pittsburgh, PA). All single proteins used as AF4 standards were purchased from Sigma-Aldrich (St. Luis, MO). Taq 5X master mix was purchased from NewEngland Biolabs.

[0085] Serum samples. The serum samples used for exosome extraction and isolat...

Embodiment 2

[0121] Microchip manufacturing. Briefly, as Figure 13 Fabrication of microchips is generally shown in . A microchip platform was fabricated by bonding together a 3" x 1" glass slide (0.5 mm thick) and a cured PDMS substrate. To fabricate the PDMS substrates, a total of three hosts were prepared. First, a first body containing only channel features, Body-1, was fabricated by an open process from thiolene-based optical adhesive NOA81. In this method, by using a collimated UV light source (365nm, ~8.3mW / cm 2 ) for 5 seconds to pre-cure NOA81 between the glass slide (plasma-treated) and the PDMS bench. The thickness of Body-1 was determined by a spacer (~400 μm) placed between the glass slide and the PDMS stage, and the features were defined by a photomask. After a brief UV exposure, the slide was slowly removed from the PDMS stage with pre-cured NOA81 based channel features on the surface. Unexposed adhesive was removed using a syringe by sequential flushing with ethanol, a...

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Abstract

The disclosure provides methods for rapid fractionation of circulating RNAs based on the type of carriers they locate in. The disclosure further provides that the methods of the disclosure can be used for diagnosing a disorder in a subject by identifying specific microRNA biomarkers associated with that disorder.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C. §119 to Provisional Application No. 62 / 045,503, filed September 3, 2014, the disclosure of which is incorporated herein by reference. [0003] government license [0004] This invention was made with government support under Grant Numbers CHE-1057113 and DGE-0813967 awarded by the National Science Foundation. The government has certain rights in this invention. technical field [0005] The present disclosure provides methods for rapid fractionation of circulating microRNAs, viral RNAs, and long non-coding RNAs (lncRNAs) based on their associated carrier molecules. The present disclosure also provides that the methods of the present disclosure can be used to diagnose a disorder in a subject by identifying specific microRNA, lncRNA and viral RNA markers associated with the disorder and specific vectors. Background technique [0006] Circulating microRNAs have been consi...

Claims

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Application Information

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IPC IPC(8): G01N30/00C12Q1/68C07H21/02C12N15/113
CPCG01N30/00C12N2310/141G01N2030/003C12Q1/6886C12Q2600/178G01N30/0005C12Q1/68
Inventor 钟文婉J·艾诗K·弗拉克
Owner RGT UNIV OF CALIFORNIA
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