Method and substances for isolating miRNAs

A method for isolating a microRNA of interest (42) from a sample comprising the microRNA of interest (42) according to FIG. 1 through FIG. 6;

• the method comprising:
• a) providing a sample comprising the microRNA of interest (42);
• b) providing a capture probe (10) comprising:
• i) a first adapter segment (12) having a first adapter segment sequence, the first adapter segment comprising a 3′ end (18) and a 5′ end (20);
• ii) a second adapter segment (14) having a second adapter segment sequence, the second adapter segment comprising a 3′ end (30) and a 5′ end (32);
• and
• iii) a microRNA binding segment (16) having a microRNA binding segment sequence;
• where the microRNA binding segment (16) is substantially complementary to, and capable of hybridizing to, one or more than one microRNA of interest (42) by Watson-Crick base pairing;
• where the 5′ end of the first adapter segment (20) is connected to the 3′ end of the microRNA binding segment (22);
• and
• where the 3′ end of the second adapter segment (30) is connected to the 5′ end of the microRNA binding segment (32);
• c) providing a first linker (48), wherein the first linker has a first linker sequence, comprises a 3′ end (52) and a 5′ end (54), and is substantially complementary to, and capable of hybridizing to, the first adapter segment (12) of the capture probe (10);
• and wherein the 5′ end of first linker (54) comprises one or more than one residue that extends beyond the 3′ end of the first adapter segment (18) after the first linker hybridizes to the first adapter segment;
• d) providing a second linker (50), wherein the second linker has a second linker sequence, comprises a 3′ end (56) and a 5′ end (58), and is substantially complementary to, and capable of hybridizing to, the second adapter segment (14) of the capture probe (10);
• e) combining the sample, the capture probe (10), the first linker (48) and the second linker (50) in a solution;
• f) allowing the first linker (48) to hybridize with the first adapter segment (12), the microRNA of interest (42) to hybridize with the microRNA binding segment (16), and the second linker (50) to hybridize with the second adapter segment (14);
• g) ligating the 3′ end of the first linker (52) to the 5′ end of the microRNA of interest (46), and ligating the 3′ end of the microRNA of interest (44) to the 5′ end of the second linker (58), 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 (48)—microRNA of interest (42)—second linker (50)) (62) and that is hybridized to the capture probe (10);
• h) dehybridizing the capture probe (10) from the strand of the ligated first linker microRNA of interest-second linker (62);
• and
• i) purifying the ligated first linker—microRNA of interest-second linker (62), thereby purifying the microRNA of interest (42).