| 主权项 |
1. A method for isolating a microRNA of interest comprising a 3′ end and a 5′ end from a sample comprising the microRNA of interest according to FIG. 1 through FIG. 6; the method comprising:
a) providing a sample comprising the microRNA of interest; b) providing a capture probe comprising:
i) a first adapter segment having a first adapter segment sequence, the first adapter segment comprising a 3′ end and a 5′ end;ii) a second adapter segment having a second adapter segment sequence, the second adapter segment comprising a 3′ end and a 5′ end; andiii) a microRNA binding segment having a microRNA binding segment sequence, the microRNA binding segment comprising a 3′ end and a 5′ end; where the microRNA binding segment is substantially complementary to, and capable of hybridizing to, one or more than one microRNA of interest by Watson-Crick base pairing; where the 5′ end of the first adapter segment is connected to the 3′ end of the microRNA binding segment; and where the 3′ end of the second adapter segment is connected to the 5′ end of the microRNA binding segment; c) providing a first linker comprising between 6 and 50 residues, where the first linker has a first linker sequence, comprises a 3′ end and a 5′ end, and is substantially complementary to, and capable of hybridizing to, the first adapter segment of the capture probe; d) providing a second linker comprising between 6 and 50 residues, where the second linker has a second linker sequence, comprises a 3′ end and a 5′ end, and is substantially complementary to, and capable of hybridizing to the second adapter segment of the capture probe; e) combining the sample, the capture probe, the first linker and the second linker in a solution; f) allowing the first linker to hybridize with the first adapter segment, the microRNA of interest to hybridize with the microRNA binding segment, and the second linker to hybridize with the second adapter segment; g) ligating the 3′ end of the first linker to the 5′ end of the microRNA of interest, and ligating the 3′ end of the microRNA of interest to the 5′ end of the second linker, thereby producing a complex defined as a strand of first linker, microRNA of interest and second linker that have been ligated together (ligated first linker- microRNA of interest-second linker) and that is hybridized to the capture probe; h) dehybridizing the capture probe from the strand of the ligated first linker-microRNA of interest-second linker; i) purifying the ligated first linker- microRNA of interest-second linker, thereby purifying the microRNA of interest; and j) sequencing the microRNA of interest portion of the strand of the ligated first linker-microRNA of interest-second linker. |
