System for determining a health status of a tissue of interest

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 method comprising:
 (a) obtaining from a pregnant subject a whole blood sample comprising a mixture of maternally-derived and fetal-derived nucleic acids;   (b) subjecting at least a portion of said whole blood sample to centrifugation in a first container, thereby separating said at least said portion of said whole blood sample into a first layer comprising plasma and a second layer comprising blood cells;   (c) transferring, from said first container to a second container, at least a portion of said first layer without transferring said second layer, thereby producing a cell-free plasma sample;   (d) isolating cell-free ribonucleic acid (RNA) molecules from said cell-free plasma sample, wherein said cell-free RNA molecules are from said pregnant subject;   (e) reverse transcribing at least a portion of said cell-free RNA molecules into complementary deoxyribonucleic acid (cDNA) molecules;   (f) amplifying at least a portion of said cDNA molecules, thereby producing amplified DNA molecules; and   (g) sequencing at least a portion of said amplified DNA molecules to determine levels of a set of pregnancy-related RNA markers.   
     
     
         2 . The method of  claim 1 , wherein said centrifugation enriches fetal-derived or placenta-derived nucleic acid molecules from said mixture. 
     
     
         3 . The method of  claim 1 , further comprising performing a first centrifugation of said whole blood sample, and isolating a first supernatant therefrom; and performing a second centrifugation of said first supernatant, and isolating a second supernatant therefrom. 
     
     
         4 . The method of  claim 1 , wherein (f) further comprises performing targeted amplification of a set of genes. 
     
     
         5 . The method of  claim 1 , wherein (f) further comprises performing unbiased amplification. 
     
     
         6 . The method of  claim 1 , wherein said pregnant subject is in a first trimester of pregnancy. 
     
     
         7 . The method of  claim 1 , wherein said pregnant subject is in a second trimester of pregnancy. 
     
     
         8 . The method of  claim 1 , wherein said pregnant subject is in a third trimester of pregnancy. 
     
     
         9 . The method of  claim 1 , further comprising:
 (h) comparing said levels of said set of pregnancy-related RNA markers determined in (g) to reference levels of said set of pregnancy-related RNA markers; and   (i) determining that said pregnant subject has, or is at an elevated risk of having, a pregnancy complication, based at least in part on said comparing in (h).   
     
     
         10 . The method of  claim 9 , wherein said pregnancy complication comprises pre-eclampsia. 
     
     
         11 . The method of  claim 9 , wherein said set of pregnancy-related RNA markers comprises RNA markers corresponding to tissue-specific differentially expressed genes. 
     
     
         12 . The method of  claim 11 , wherein said tissue-specific differentially expressed genes comprise maternal-specific genes. 
     
     
         13 . The method of  claim 11 , wherein said tissue-specific differentially expressed genes comprise placental-specific genes. 
     
     
         14 . The method of  claim 11 , wherein said tissue-specific differentially expressed genes comprise fetal-specific genes. 
     
     
         15 . The method of  claim 14 , wherein said fetal-specific genes comprise genes corresponding to a fetal tissue type selected from the group consisting of: brain, liver, thymus, and lung. 
     
     
         16 . The method of  claim 9 , further comprising providing a clinical intervention for said pregnant subject having, or at said elevated risk of having, said pregnancy complication, based on said determining in (i). 
     
     
         17 . The method of  claim 16 , wherein said clinical intervention comprises a drug treatment. 
     
     
         18 . The method of  claim 9 , wherein said reference levels correspond to a set of pregnancy-related RNA markers from non-pregnant subjects. 
     
     
         19 . The method of  claim 9 , wherein said reference levels correspond to a set of pregnancy-related RNA markers from pregnant subjects. 
     
     
         20 . The method of  claim 9 , wherein comparing said levels of said set of pregnancy-related RNA markers to said reference levels comprises determining a difference between said levels of said set of pregnancy-related RNA markers and said reference levels; and using said difference to determine that said pregnant subject has, or is at said elevated risk of having, said pregnancy complication. 
     
     
         21 . The method of  claim 20 , further comprising determining a level of fold change in quantitative polymerase chain reaction (qPCR) measurements based at least in part on said levels of said set of pregnancy-related RNA markers relative to said reference levels to determine said difference. 
     
     
         22 . The method of  claim 9 , wherein (h) further comprises determining relative contributions of distinct fetal or placental tissue types; and determining that said pregnant subject has, or is at said elevated risk of having, said pregnancy complication, based at least in part on said relative contributions of said distinct fetal or placental tissue types. 
     
     
         23 . The method of  claim 9 , further comprising performing (a) to (i) for whole blood samples obtained from said pregnant subject at two or more different time points; and comparing said levels of said sets of pregnancy-related RNA markers to each other to determine that said pregnant subject has, or is at said elevated risk of having, said pregnancy complication. 
     
     
         24 . The method of  claim 9 , further comprising monitoring said pregnant subject for said pregnancy complication.