Use of circulating cell-free RNA for diagnosis and/or monitoring cancer
The invention provides compositions, methods, and systems for using cell-free RNA for improved detection of rare cells and/or species that are useful for diagnosing and/or monitoring cancer. The invention also provides for compositions, methods, and systems for early detection of cells that are resistant and/or developing resistance to cancer therapies.
1. A method for detecting PD-L1 mRNA in a blood sample from an individual, the method comprising:
a. isolating cfRNA from the blood sample on a solid support, wherein the blood sample has been interacted with RNA stabilizer;
b. digesting DNA from the biological sample while the cfRNA is on the solid support;
c. eluting cfRNA at least once from the solid support;
d. reverse transcribing the cfRNA to cDNA;
e. reacting the cDNA with at least one primer that is specific for
i. a PD-L1 gene mutation, or
ii. a PD-L1 fusion transcript, or
iii. PD-L1 gene expression.
2. The method of claim 1 wherein the eluate of step (c) is passed over the same column for a dual elution prior to the reverse transcribing the cfRNA to cDNA.
3. The method of claim 1 wherein the individual has or is suspected of having cancer.
4. The method of claim 3 wherein the plasma is processed within 7 days of interacting with the RNA stabilizer.
5. The method of claim 1 wherein random hexamers are used in step (d) to reverse transcribe the RNA to cDNA.
6. The method of claim 1 , further comprising sequencing the cDNA.
7. The method of claim 6 , wherein the sequencing comprises polymerase chain reaction (PCR)-based sequencing.
8. The method of claim 7 , wherein the PCR-based sequencing is next generation sequencing.