Methods for sequencing cell-free RNA
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.
1 . A method comprising:
(a) obtaining a whole blood sample from a subject;
(b) extracting cell-free ribonucleic acid (RNA) from plasma of the whole blood sample of the subject;
(c) converting the cell-free RNA into complementary deoxyribonucleic acid (cDNA) thereby producing sample cDNA;
(d) amplifying the sample cDNA to produce amplified DNA;
(e) sequencing the amplified DNA to produce sequence information;
(f) quantitating the sequence information to determine a level of the sample cDNA that encodes one or more RNA transcripts comprising brain-specific RNA transcripts selected from the group consisting of APP, PSD3, MOBP, MAG, SLC2A1, TCF7L2, CDH22, CNTF and PAQR6;
and
(g) determining that the subject has Alzheimer's disease, based on the quantified levels of the one or more RNA transcripts.
2 . The method of claim 1 , wherein (b) further comprises extracting total RNA from the plasma of the whole blood sample.
3 . The method of claim 2 , wherein extracting the total RNA comprises contacting the plasma of the whole blood 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 . The method of claim 1 , wherein the whole blood sample has a volume of 7 milliliters to 15 milliliters.
8 . The method of claim 1 , wherein the amplifying comprises polymerase chain reaction (PCR).
9 . The method of claim 8 , wherein the PCR comprises indiscriminate PCR.
10 . The method of claim 1 , wherein the sequencing comprises whole transcriptome sequencing.
11 . The method of claim 1 , further comprising performing microarray analysis of the sample cDNA.
12 . The method of claim 1 , further comprising performing quantitative polymerase chain reaction (PCR) analysis of the sample cDNA.