Methods for RNA analysis

A method for analyzing a population of RNA molecules comprising the following steps:

• (i) providing a population of RNA molecules, wherein the population of RNA molecules comprises at least two different types of RNA molecules, wherein the different types of RNA molecules comprise an identical target sequence;
• (ii) providing a conjugate comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the sequence of said oligonucleotide is complementary to the target sequence;
• (iii) cleaving the population of RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugate provided in step (ii) under conditions allowing the hybridization of said oligonucleotide to said target sequence and the cleavage of the RNA molecules;
• and
• (iv) determining a physical property of the RNA molecules in the population by analyzing one or more of the RNA fragments obtained in step (iii),
• wherein the RNA molecules are mRNA molecules and each comprise a 5′ cap structure and/or a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecules comprises one or more temperature shifts, wherein step (iii) of the method is as follows:
• (iii) cleaving the RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugates provided in step (ii) at a first temperature between about 5° C. and about 50° C., and at a second temperature between about 70° C. and about 90° C.;
• followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).

A method for analyzing a population of RNA molecules comprising the following steps:

• (i) providing a population of RNA molecules, wherein the population of RNA molecules comprises at least two different types of RNA molecules, wherein the different types of RNA molecules comprise different target sequences;
• (ii) providing at least two conjugates comprised of a chemical moiety with RNA cleaving activity and an oligonucleotide, wherein the oligonucleotide sequence of each conjugate is complementary to one of the different target sequences;
• (iii) cleaving the population of RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the at least two conjugates provided in step (ii) under conditions allowing the hybridization of said oligonucleotides to said target sequences and the cleavage of the RNA molecules;
• and
• (iv) determining a physical property of the RNA molecules in the population by analyzing one or more of the RNA fragments obtained in step (iii),
• wherein the RNA molecules are mRNA molecules and each comprise a 5′ cap structure and/or a PolyA sequence, wherein the conditions allowing the hybridization and the cleavage of the RNA molecules comprises one or more temperature shifts, wherein step (iii) of the method is as follows:
• (iii) cleaving the RNA molecules provided in step (i) to obtain RNA fragments by contacting the RNA molecules with the conjugates provided in step (ii) at a first temperature between about 5° C. and about 50° C., and at a second temperature between about 70° C. and about 90° C.;
• followed by a step of repeating the above step (iii) at least once, wherein this step precedes step (iv).