Methods and kits for extracting nucleic acids from paraffin embedded samples
Inventors
Qin, Jian • Chen, Peilin • Ramakrishnan, Ramesh
Assignees
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Publication Number
US-11078476-B2
Publication Date
2021-08-03
Expiration Date
Abstract
This invention provides a technology for isolating nucleic acids from wax-embedded samples that is superior to the current state of the art. Standard protocols with this objective typically comprise dissolving the wax-embedded sample in an organic solvent, extracting nucleic acids from the organic solvent into an aqueous buffer, and isolating the nucleic acids from the aqueous buffer. The technology described here includes using hexadecane as the solvent to dissolve the sample, precipitating and washing the extracted nucleic acids, and dissolving the nucleic acids in a lysis buffer that includes NP40 and SDS. By implementing the reagents and techniques described in this disclosure, the user can obtain a product that has better yield, less degradation, and contains more unique mRNA transcripts for subsequent sequencing and analysis.
Core Innovation
The document describes methods for extracting nucleic acids from a wax-embedded biological sample by combining the sample with a wax-solubilizing organic solvent comprising a hydrocarbon of at least 10 carbon atoms to form a mixture. An alcohol is added to form a precipitate containing nucleic acids, and the organic solvent and the alcohol are separated from the precipitate. The precipitate is then combined with a lysis buffer to dissolve at least some of the precipitated nucleic acids to form a lysis solution.
The nucleic acids in the lysis solution are combined with beads adapted to bind nucleic acids, and nucleic acids are recovered from the first aliquot of beads. The recovered nucleic acids are treated in a buffer containing DNase to remove deoxyribonucleic acids, and RNA is isolated from the DNase-containing buffer using a second aliquot of beads adapted to bind nucleic acids. The lysis buffer contains a protease and one or more detergents that include nonyl-phenoxypolyethoxylethanol (NP40), sodium dodecyl sulfate (SDS), or a combination of both NP40 and SDS.
Additionally, the document presents an improvement to a protocol for isolating nucleic acids from wax-embedded biological samples, where the improvement uses a saturated hydrocarbon comprising at least 10 carbon atoms as the organic solvent and causes nucleic acids in the organic solvent to form a precipitate that is washed to remove the organic solvent and alcohol. The improvement further includes NP40 and an RNA inhibitor in the aqueous buffer, treating the nucleic acids with DNase, and isolating nucleic acids both before and after the DNase treatment using beads.
Claims Coverage
The independent claims cover three related inventive methods: extracting nucleic acids from wax-embedded biological samples; improving recovery within a nucleic-acid isolation protocol from wax-embedded samples; and extracting ribonucleic acids (RNA) with staged DNase treatment and separate bead capture. Across the independent claims, the coverage emphasizes precipitation after wax-solubilization, bead-based nucleic-acid recovery, and DNase-mediated removal of deoxyribonucleic acids in a workflow that uses NP40 and SDS, and a protease, in the lysis buffer.
Wax-solubilizing hydrocarbon and alcohol-driven precipitation
Combining the biological sample with a wax-solubilizing organic solvent comprising a hydrocarbon of at least 10 carbon atoms to form a mixture, adding an alcohol to the mixture, and forming a precipitate that contains the nucleic acids; then separating the organic solvent and the alcohol from the precipitate.
Lysis buffer with protease and NP40 and/or SDS
Combining the precipitate with a lysis buffer so as to dissolve at least some of the precipitated nucleic acids to form a lysis solution, where the lysis buffer contains a protease and one or more detergents that include NP40, SDS, or a combination of both NP40 and SDS.
Bead binding and recovery of nucleic acids
Combining the lysis solution with a first aliquot of beads adapted to bind nucleic acids and recovering nucleic acids derived from the biological sample from the first aliquot of beads.
DNase treatment and RNA isolation using a second bead aliquot
Treating the recovered nucleic acids in a buffer that contains DNase to remove deoxyribonucleic acids, and thereafter isolating RNA from the buffer containing DNase using a second aliquot of beads adapted to bind nucleic acids.
Saturated hydrocarbon precipitation with NP40 and RNA inhibitor and DNase plus bead isolation before and after DNase
Using a saturated hydrocarbon comprising at least 10 carbon atoms as the organic solvent; causing the nucleic acids in the organic solvent to form a precipitate that is then washed to remove the organic solvent and alcohol; including NP40 and an RNA inhibitor in the aqueous buffer; treating the nucleic acids with DNase; and isolating the nucleic acids both before and after the DNase treatment using beads.
RNA extraction workflow with NP40, SDS, proteinase K, and staged DNase capture
Combining the washed precipitate with a lysis buffer that contains NP40, SDS, and proteinase K to dissolve nucleic acids to form a lysis solution; combining the lysis solution with a first aliquot of magnetic beads adapted to reversibly bind nucleic acids and recovering nucleic acids; treating the recovered nucleic acids in a buffer that contains DNase to remove deoxyribonucleic acids; combining the lysis solution containing the DNase treated nucleic acids with a second aliquot of magnetic beads adapted to reversibly bind nucleic acids; and recovering nucleic acids derived from the biological sample from the second aliquot of beads, thereby obtaining ribonucleic acids (RNA) from the wax-embedded biological sample.
Overall, the independent claims require a wax-solubilizing saturated hydrocarbon (hydrocarbon of at least 10 carbon atoms), alcohol-driven nucleic-acid precipitation followed by washing, bead-based nucleic-acid recovery, and DNase treatment to remove deoxyribonucleic acids, with RNA obtained via staged recovery using NP40 and SDS, and a protease such as proteinase K, in the lysis buffer. The improvement claim further specifies NP40 together with an RNA inhibitor in the aqueous buffer and isolation before and after DNase using beads.
Stated Advantages
Improved yield, including increased RNA recovery compared with commercial kits in the reported comparisons.
Reduced degradation/fragmentation, including higher DV200 compared with commercial kits in the reported comparisons.
Increased numbers of unique mRNA transcripts/genes detected by qPCR and RNA-seq compared with commercial kits in the reported comparisons.
Documented Applications
qPCR and RNA-seq gene-expression analysis from RNA recovered using the described wax-embedded nucleic-acid extraction workflow.
Performance comparison of the described extraction methods against commercial kits, including measurement of DV200 and qPCR/RNA-seq detection outputs.
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