Multiplexed signal amplified FISH via splinted ligation amplification and sequencing
The present invention relates to a method for amplifying at least one target RNA in a fixed and, optionally, expanded biological sample. In an embodiment of the invention, the method comprises incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA; ligating the polynucleotide pair using a ligase; and amplifying the ligation product. The invention further provides methods for detecting and optionally quantifying and/or sequencing the amplification product. As the method comprises hybridizing polynucleotide pairs to a target RNA in a fixed biological sample, the target RNA can be hybridized in situ.
1. A method for amplifying one or more target RNAs in a fixed biological sample comprising:
(a) contacting the sample with a small molecule linker or a nucleic acid adaptor comprising a binding moiety and an anchor, wherein the binding moiety binds to target nucleic acids in the sample; and wherein the anchor comprises a polymerizable moiety;
(b) permeating the sample with a composition comprising precursors of a swellable material; and
(c) initiating polymerization of the precursors of the swellable material to form a swellable material, wherein the swellable material is bound to the small molecule linker or a nucleic acid adaptor to form a sample-swellable material complex;
(d) incubating the sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA wherein the polynucleotides hybridize to the target RNA;
(e) ligating the polynucleotide pair using a ligase; and
(f) amplifying the ligation product.
2. The method of claim 1 , further comprising expanding the sample.
3. The method of claim 2 , wherein expanding the sample comprises adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex.
4. The method of claim 2 , wherein the biological sample is expanded prior to step (f) or post step (f).
5. The method of claim 1 , wherein the swellable material is a polyelectrolyte gel.
6. The method of claim 1 , wherein the pair of polynucleotides is hybridized to the target RNA in situ.
7. The method of claim 1 , further comprising the step of sequencing the amplified ligation product within the sample.
8. A method for detecting one or more target RNAs in a fixed biological sample comprising:
(a) contacting the sample with a small molecule linker or a nucleic acid adaptor comprising a binding moiety and an anchor, wherein the binding moiety binds to target nucleic acids in the sample; and wherein the anchor comprises a polymerizable moiety;
(b) permeating the sample with a composition comprising precursors of a swellable material; and
(c) initiating polymerization of the precursors of the swellable material to form a swellable material, wherein the swellable material is bound to the small molecule linker or a nucleic acid adaptor to form a sample-swellable material complex;
(d) incubating the sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA wherein the polynucleotides hybridize to the target RNA;
(e) ligating the polynucleotide pair using a ligase;
(f) amplifying the ligation product; and
(g) detecting the amplified product.
9. The method of claim 8 , further comprising expanding the sample.
10. The method of claim 9 , wherein expanding the sample comprises adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex.
11. The method of claim 9 , wherein the biological sample is expanded prior to step (f) or post step (f).
12. The method of claim 8 , wherein the swellable material is a polyelectrolyte gel.
13. The method of claim 8 , wherein the pair of polynucleotides is hybridized to the target RNA in situ.
14. The method of claim 8 , further comprising the step of localizing the amplified product within the fixed biological sample.
15. The method of claim 8 , further comprising the step of sequencing the amplified ligation product within the fixed biological sample.