Method of determining a ratio of RNA species in a sample

摘要

A method of amplifying DNA from RNA in a sample by using circular RNA is provided.

主权项

1. A method of determining a ratio of the amounts of two or more RNAs in a sample, the method comprising:
a) providing a sample comprising two or more different RNAs; b) incubating the sample in the presence of: an enzyme that converts the 5′-cap structure into 5′-monophosphate to convert RNA having a 5′-cap structure in the sample into RNA having 5′-monophosphate; an enzyme that phosphorylates 5′-OH to convert RNA having 5-OH in the sample into RNA having 5′-monophosphate; and an enzyme that dephosphorylates a 3′-end to convert RNA having a 3′-phosphate group in the sample into RNA having 3′-OH, thereby producing a mixture comprising two or more different RNAs having a 5′-monophosphate and a 3′-OH in the sample; c) subsequently incubating the mixture in the presence of an RNA ligase after step b), thereby producing circular RNAs from the two or more different RNAs having a 5′-monophosphate and a 3′-OH, wherein the RNA ligase catalyzes intramolecular ligation of each of the two or more different RNAs having a 5′-monophosphate and a 3′-OH; d) incubating the circular RNAs in the presence of an RNA-dependent DNA polymerase, thereby producing DNAs from the circular RNAs; e) from the DNAs, identifying sequences of DNA molecules that correspond to sequences of the two or more different RNAs in the sample and determining the amounts of the DNA molecules that correspond to the two or more different RNAs in the sample; and f) determining a ratio of the amounts of the DNA molecules that correspond to the two or more different RNAs in the sample, wherein the ratio of amounts of the DNA molecules correlates with the ratio of the amounts of the two or more different RNAs in the sample.