The present invention is directed to compositions, methods and kits useful for the generation of nucleic acids from RNA templated and further nucleic acid amplification and detection. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase polymerase chain reaction (RT-PCR). Provided are compositions and methods for improved amplification of nucleic acid molecules in a two-step, addition only RT-PCR procedure. The invention thus facilitates the rapid and efficient amplification of nucleic acid molecules and the detection and quantitation of RNA molecules, and is useful for a variety of research, industrial, medical and forensic purposes.

Provided herein are methods for de-crosslinking fixed biological samples (e.g., fixed biological samples including aminal crosslinks). The compositions and methods disclosed can de-crosslink oligonucleotides (e.g., DNA or RNA) or proteins from fixed biological samples (e.g., fixed biological samples with aminal crosslinks), wherein the de-crosslinked biological sample is compatible with and can be used in spatial gene expression analysis.

Provided herein are methods for de-crosslinking fixed biological samples (e.g., fixed biological samples including aminal crosslinks). The compositions and methods disclosed can de-crosslink oligonucleotides (e.g., DNA or RNA) or proteins from fixed biological samples (e.g., fixed biological samples with aminal crosslinks), wherein the de-crosslinked biological sample is compatible with and can be used in spatial gene expression analysis.

Provided herein are engineered variants of archaeal, prokaryotic, and eukaryotic polymerases that exhibit enhanced thermostability, enhanced incorporation of 3′ modified nucleotides, and improved uracil-tolerance, in polymerase-catalyzed nucleotide extension reactions relative to wild type polymerase enzymes. Also provided are uses of the engineered polymerases for forming complexed polymerases, forming binding complexes and forming ternary complexes, and uses for conducting nucleic acid sequencing reactions.

Provided herein are engineered variants of archaeal, prokaryotic, and eukaryotic polymerases that exhibit enhanced thermostability, enhanced incorporation of 3′ modified nucleotides, and improved uracil-tolerance, in polymerase-catalyzed nucleotide extension reactions relative to wild type polymerase enzymes. Also provided are uses of the engineered polymerases for forming complexed polymerases, forming binding complexes and forming ternary complexes, and uses for conducting nucleic acid sequencing reactions.

A method of enriching nucleic acid, including mixing a sample with a solid phase extraction material; and separating the solid phase extraction material; wherein the solid phase extraction material is glass beads or magnetic beads modified with reduced graphene oxide. Also disclosed is a method of detecting nucleic acid, including mixing a nucleic acid sample enriched by the method above with a probe; and amplifying and detecting an amplification product by electrophoresis.

A method of enriching nucleic acid, including mixing a sample with a solid phase extraction material; and separating the solid phase extraction material; wherein the solid phase extraction material is glass beads or magnetic beads modified with reduced graphene oxide. Also disclosed is a method of detecting nucleic acid, including mixing a nucleic acid sample enriched by the method above with a probe; and amplifying and detecting an amplification product by electrophoresis.

Capture mixtures and activated capture mixtures are provided that are useful for nucleic acid separation and purification are provided. The mixtures comprise lithium lauryl sulfate, lithium hydroxide, a zwitterionic sulfonic acid buffering agent, and optionally, proteinase K, capture probes comprising a first specific binding partner (SBP), and a second specific binding partner immobilized to a solid support. Related combinations, methods, uses, and kits, are also provided.

Capture mixtures and activated capture mixtures are provided that are useful for nucleic acid separation and purification are provided. The mixtures comprise lithium lauryl sulfate, lithium hydroxide, a zwitterionic sulfonic acid buffering agent, and optionally, proteinase K, capture probes comprising a first specific binding partner (SBP), and a second specific binding partner immobilized to a solid support. Related combinations, methods, uses, and kits, are also provided.

An ultraviolet (UV) absorbance assay for measuring the concentration of large RNA molecules such as mRNA in suspensions comprising RNA-lipid nanoparticles (RNA-LNPs) is described.