Methods for profiling and quantitating cell-free rna

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.

What is claimed is: 
     
         1 . A computer system for determining a health state of a tissue of interest in a subject, comprising:
 a) at least one processor;   b) a system memory; and   c) one or more computer-readable storage media having stored thereon a set of encoded, computer-executable instructions that cause the computer system to:   i) store data representing a plurality of sample quantities of a plurality of cell-free messenger RNAs in a test biological sample of a test subject;   ii) calculate a relative contribution of a pathway of interest to a cell-free transcriptome of the biological sample based on the sample quantities;   iii) calculate a difference between the relative contribution and a reference relative contribution of the pathway in a reference sample of a reference subject, wherein the reference subject is characterized by a known health status; and   iv) assign a health status to the test subject based on a presence or absence of a statistically significant difference between the relative contribution and the reference relative contribution.   
     
     
         2 . The computer system of  claim 1 , wherein the pathway is involved in at least one of apoptosis, cancer, an autoimmune disorder, a metabolic disease, and inflammation. 
     
     
         3 . A system, comprising:
 a) a computer processor;   b) a computer-readable medium coupled with the computer processor, the computer-readable medium storing a plurality of instructions wherein, when implemented by the processor, said instructions direct the processor to perform a plurality of logical steps comprising:   i) receiving a collection of data from a nucleic acid quantification dataset, wherein the nucleic acid quantification dataset comprises a plurality of sample quantities of a plurality of cell-free messenger RNAs from a plurality of tissues in a test biological sample of a test subject;   ii) deconvoluting reference relative contributions of the plurality of tissues to a reference cell free transcriptome in a reference sample of a reference subject;   iii) calculating relative contributions of the plurality of tissues in the test biological sample to a cell-free transcriptome based on the sample quantities; and   iv) calculating a difference between the relative contributions and the reference relative contributions; and   iv) determining whether the health state of the tissue of interest in the subject differs or does not differ from the known reference health state based on the principle component analysis.   
     
     
         4 . The system of  claim 3 , wherein the said instructions direct the processor to perform an additional logical step comprising assigning a health status to the test subject based on a presence or absence of a statistically significant difference between the relative contribution and the reference relative contribution. 
     
     
         5 . The system of  claim 3 , wherein calculating a difference between the relative contributions and the reference relative contributions comprises performing a principle component analysis on the relative contributions and reference relative contributions of the reference subject. 
     
     
         6 . The system of  claim 3 , wherein deconvoluting comprises identifying a panel of tissue-specific transcripts; determining total RNA in the test biological sample; and assessing the total RNA against the panel of tissue-specific transcripts, wherein the total RNA is considered a summation the tissue-specific transcripts. 
     
     
         7 . The system of  claim 6 , wherein identifying a panel of tissue-specific transcripts comprises applying a template-matching algorithm to a first gene expression database and a second gene expression database. 
     
     
         8 . The system of  claim 7 , wherein applying the template-matching algorithm comprises regression of gene expression across different tissue type. 
     
     
         9 . The system of  claim 3 , wherein the computer-readable medium filters for a correlation coefficient of about 0.9. 
     
     
         10 . The system of  claim 3 , wherein the computer-readable medium filters for a p-value of less than 0.0001. 
     
     
         11 . The system of  claim 3 , wherein the computer-readable medium is in a form selected from RAM, ROM, EEPROM, flash memory, CD-ROM, DVD, magnetic cassettes, magnetic tape, and magnetic disk storage. 
     
     
         12 . The system of  claim 3 , wherein receiving the plurality of sample quantities comprises receiving microarray probe intensities and the computer-readable medium comprises a raw microarray algorithm (RMA) to process microarray data. 
     
     
         13 . The system of  claim 12 , wherein the processor converts microarray probe intensities to log scale. 
     
     
         14 . The system of  claim 2 , wherein receiving the plurality of sample quantities comprises receiving sequencing data. 
     
     
         15 . The system of  claim 14 , wherein the computer-readable medium obtains reads per kilobase per million map reads (RPKM) values from the sample quantities. 
     
     
         16 . The system of  claim 3 , wherein receiving the plurality of sample quantities comprises receiving RNA-Seq data. 
     
     
         17 . The system of  claim 3 , wherein receiving the plurality of sample quantities comprises receiving Q-PCR data. 
     
     
         18 . The system of  claim 3 , wherein receiving the plurality of sample quantities comprises receiving microarray probe intensities and RNA-Seq data. 
     
     
         19 . The system of  claim 18 , wherein the computer-executable instructions comprise instructions for calculating correlation coefficients between microarray probe intensities and RNA-Seq data. 
     
     
         20 . A computer readable medium that distinguishes between a health state of a tissue of interest in a test subject and a known reference health state of the tissue of interest in a reference subject by performing a principle component analysis on
 a) a relative contribution of the tissue of interest to a cell-free transcriptome in a test sample of the test subject, and   b) a reference relative contribution of the tissue of interest to a reference cell-free transcriptome in a reference test sample of the reference subject.