Methods and compositions for determining nucleic acid degradation
Described herein are methods, systems, compositions and kits to enable determination of a level of degradation of nucleic acids in a sample. For example, the determination of the amount of degradation of RNA in a sample can be accomplished by labeling one or both of the intact 5′- and/or 3′-ends of an mRNA molecule by taking advantage of the unique diol moiety present at these structures. Labeled nucleotides can then be partitioned into droplets and the amount of degradation can be determined by detecting label present in the partitions and making quantitative or qualitative comparisons with a reference sample. In some cases, the degree of degradation is also determined by factoring in the total concentration or quantity of RNA in the sample.
1. A method of detecting degradation of mRNA, the method comprising:
(a) contacting a sample comprising mRNA with at least one agent to covalently attach an oligonucleotide label to a first end of RNA molecules having a first end structure and to covalently attach an oligonucleotide label to a second end of mRNA molecules having a second end structure;
(b) separating the sample into a plurality of spatially isolated partitions;
(c) enumerating a first number of spatially isolated partitions comprising an oligonucleotide label covalently attached to the first end of an mRNA molecule and an oligonucleotide label covalently attached to the second end of an mRNA molecule, and a second number of spatially isolated partitions comprising an oligonucleotide label covalently attached only to the first end or only to the second end of an mRNA molecule; and
(d) determining an amount of degradation for the mRNA based on the enumerating, wherein the step of determining comprises comparing the first number and the second number to one another.
2. The method of claim 1 , wherein the separating is randomized.
3. The method of claim 1 , wherein covalent attachment of an oligonucleotide label to the first end of mRNA molecules and covalent attachment of an oligonucleotide label to the second end of mRNA molecules is not sequence specific.
4. The method of claim 1 , wherein the second end of mRNA molecules having a 3′-phosphate group or a 2′-3′-linked phosphate group are not substantially labeled with an oligonucleotide label by the step of contacting.
5. The method of claim 1 , wherein the first end of mRNA molecules having a 5′-phosphate group and the second end of mRNA molecules having a 3′-hydroxyl group are substantially labeled with an oligonucleotide label by the step of contacting.
6. The method of claim 1 , wherein the at least one agent comprises a ligase.
7. The method of claim 1 , wherein the second end of mRNA molecules including a ribose diol group are substantially labeled by the step of contacting.
8. The method of claim 1 , wherein mRNA molecules comprising an intact 5′-end cap are substantially labeled by the step of contacting.
9. The method of claim 1 , wherein the spatially isolated partitions are droplets within an emulsion.
10. The method of claim 1 , wherein the determining is accomplished using an algorithm.
11. The method of claim 8 , wherein the at least one agent comprises a decapping enzyme.
12. The method of claim 6 , wherein the at least one agent comprises a T4 RNA ligase.
13. The method of claim 1 , wherein the oligonucleotide covalently attached to the second end comprises, before attachment, a 5′-adenylated end, a blocked 3′-end, and a target sequence for an amplification reaction.
14. The method of claim 13 , wherein the amplification reaction is ddPCR.
15. The method of claim 1 , further comprising enumerating a number of spatially isolated partitions comprising mRNA from a reference sample.
16. The method of claim 15 , the sample being a test sample, wherein the steps of contacting, separating, and enumerating are also performed with a reference sample, and wherein the step of determining an amount of degradation for the mRNA based on the enumerating comprises comparing the number for the test sample and the number for the reference sample with one another.
17. The method of claim 1 , further comprising determining a concentration of mRNA in the sample.
18. The method of claim 1 , wherein the oligonucleotide covalently attached to the first end has a different sequence from the oligonucleotide covalently attached to the second end.