Methods and compositions for improving removal of ribosomal RNA from biological samples
The invention generally relates to compositions for maximizing capture of affinity-labeled molecules on solid supports. The disclosed methods and compositions were developed to maximize depletion of ribosomal RNA from total RNA samples, which is useful to improve the quality of RNA preparations used for applications such as massively parallel sequencing. The RNA depletion method is based on using long affinity-labeled RNA molecules that are complementary to all or part of the target ribosomal RNAs, as subtractive hybridization probes.
1. A method for removing 18S rRNA from an RNA sample comprising:
obtaining an RNA sample from a biological source, said RNA sample containing 18S rRNA;
mixing said RNA sample with one or more RNA subtractive hybridization probes, thereby forming a mixture comprising the RNA sample and the one or more RNA subtractive hybridization probes, wherein the one or more RNA subtractive hybridization probes comprise an affinity label comprising biotin, and wherein the one or more of the RNA subtractive hybridization probes hybridize to two or more discontinuous regions of 18S rRNA in the RNA sample;
incubating the mixture under conditions effective to allow hybridization of the one or more RNA subtractive hybridization probes with the 18S rRNA in the RNA sample, thereby forming a hybridized complex comprising the one or more RNA subtractive hybridization probes and the 18S rRNA;
facilitating association of the hybridized complex with a solid support by incubating the hybridized complex with the solid support in a reaction composition, thereby forming a complex comprising the hybridized complex and the solid support, wherein the affinity label on the one or more of the RNA subtractive hybridization probes in the hybridized complex specifically binds to an affinity label recognition molecule attached to the solid support, wherein the affinity label recognition molecule comprises streptavidin or neutravidin; and
removing the 18S rRNA from the RNA sample by removing the complex comprising the hybridized complex and the solid support from the reaction composition;
wherein a combination of polyethylene glycol and magnesium ions in the reaction composition enhances removal of the complex comprising the hybridized complex and the solid support from the reaction composition, and
wherein the reaction composition consists of: polyethylene glycol at a concentration between about 2% and 25%, magnesium ions at a concentration between about 3 mM and 30 mM, and sodium chloride at a concentration about 150 mM.
2. The method of claim 1 , further comprising recovering RNA remaining in the reaction composition after the removing step.
3. The method of claim 1 , wherein one of the one or more RNA subtractive hybridization probes are derived from a 788 bp region of the 18S rRNA.
4. The method of claim 1 , wherein the one or more of RNA subtractive hybridization probes are single-stranded RNA probes.
5. A method for removing 18S rRNA from an RNA sample, comprising:
obtaining an RNA sample from a biological source;
mixing said RNA sample with one or more RNA subtractive hybridization probes, thereby forming a mixture comprising the RNA sample and the one or more RNA subtractive hybridization probes, wherein the one or more RNA subtractive hybridization probes comprise an affinity label comprising biotin, and wherein the one or more the RNA subtractive hybridization probes hybridize to two or more discontinuous regions of 18S rRNA in the RNA sample;
incubating the mixture under conditions effective to allow hybridization of the one or more RNA subtractive hybridization probes with the 18 S rRNA in the RNA sample, thereby forming a hybridized complex comprising the one or more RNA subtractive hybridization probes and the 18S rRNA;
facilitating association of the hybridized complex with a solid support by incubating the hybridized complex with the solid support in a reaction composition, thereby forming a complex comprising the hybridized complex and the solid support, wherein the affinity label on the one or more the RNA subtractive hybridization probes in the hybridized complex specifically binds to an affinity label recognition molecule attached to the solid support, wherein the affinity label recognition molecule comprises streptavidin or neutravidin; and
removing the 18S rRNA from the RNA sample by removing the complex comprising the hybridized complex and the solid support from the reaction composition; and,
wherein the reaction composition consists of: polyethylene glycol at a concentration between about 2% and 25%, magnesium ions at a concentration between about 3 mM and 30 mM, and sodium chloride at a concentration about 150 mM, and
wherein a combination of the polyethylene glycol and the magnesium ions in the reaction composition enhances a removal of the complex comprising the hybridized complex and the solid support from the reaction composition.
6. The method of claim 5 , wherein the RNA sample comprises a mixture of RNA from eukaryotic and prokaryotic cells.
7. The method of claim 5 , wherein the one or more of RNA subtractive hybridization probes are single-stranded RNA probes.