New England Biolabs, E7750S, NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat)

Globin mRNA and rRNA are highly abundant in blood samples, and their efficient and specific removal is desirable in order to reveal the biological significance of less abundant transcripts. Related Categories RNA Depletion & mRNA Enrichment,, Epitranscriptome Analysis,, RNA Library Prep for Illumina Specification Materials Required but not Supplied Magnetic rack/stand such as the NEBNext Magnetic Separation Rack (NEB #S1515) 80% Ethanol (freshly prepared) Thermocycler Thin wall 200 μl PCR tubes Bioanalyzer®, TapeStation® (Agilent Technologies, Inc.) or similar fragment analyzer and consumables Agencourt® RNAClean® XP Beads (Beckman Coulter, Inc. #A63987) FAQ Q: What species can I use the NEBNext® Globin mRNA & rRNA Depletion Kit for? Will it work for other species? A: The kit depletes globin mRNA and rRNA from human, mouse and rat total RNA. It can work in other species to the degree to which the rRNA and globin mRNA sequences are similar to that of human, mouse and rat. Q: Which globin mRNAs and rRNAs are depleted with the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat)? A: The kit depletes adult, fetal and embryonic globin mRNA (HBA 1/2, HBB, HBD, HBM, HBG1/2, HBE, HBQ1 and HBZ). Cytoplasmic (5S, 5.8S, 18S, 28S, ITS, and ETS) and mitochondrial (12S and 16S) rRNA are also depleted. Q: Can I use the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) with degraded RNA or fragmented RNA? A: Yes, this kit can be used with degraded or fragmented RNA. Q: What is the total RNA input I should use? A: The kit has been optimized for 10-1,000 ng total RNA input (100 ng minimum for fragmented or degraded RNA). For RNA-seq, we recommend using inputs higher than 100 ng to increase library complexity and reduce duplication rate. Q: Why must the RNA be free of DNA? A: Contaminating DNA can cause inaccurate RNA quantification and impede proper globin mRNA and rRNA removal. Q: What is the expected RNA yield recovered after globin mRNA and rRNA depletion? A: The percentage of RNA isolated after globin and rRNA depletion will vary among different RNA samples. Typically, 1-3% of input RNA is recovered. Q: What is the percentage of globin mRNA and rRNA left after depletion? A: The percentage of globin mRNA and rRNA left after depletion will vary by species. Typically, less than 1% globin mRNA, and between 1% (human) and 5% (mouse/rat) rRNA remains. Q: How can I determine if the globin mRNA and rRNA depletion was efficient? A: To assess depletion efficiency, design RT qPCR primers for the sample species’ globin mRNA and rRNA, and primers for a housekeeping gene. Compare globin mRNA and rRNA content before and after depletion. Q: I am only interested in coding RNA. Can I use the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) to remove globin mRNA from previously enriched mRNA. A: Yes. The kit can be used following poly (A) mRNA enrichment (e.g. using the NEBNext Poly(A) mRNA Magnetic Isolation Module, NEB #E7490), so that the final depleted RNA contains only mRNA of interest and not non-coding RNA. Q: Does the NEBNext® Globin mRNA & rRNA Depletion Kit (Human/Mouse/Rat) work for ribosomal footprinting or profiling applications? A: The protocol provided with this kit has not been validated for ribosomal footprinting or profiling. Q: Can I use Agencourt® AMPure® XP Magnetic Beads instead of NEBNext® RNA Sample Purification Beads (RNAClean® XP Magnetic Beads)? A: Yes, it is possible to use AMPure XP Beads. However, we recommend using the RNAClean XP Beads/ NEBNext® RNA Sample Purification Beads (supplied in NEB #E7755) as these beads have been manufactured and tested to eliminate RNase contamination. A version of the kit includes these beads: NEBNext® Globin mRNA and rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads (NEB #E7755). Q: Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads? A: Although we recommend using RNAClean® XP beads NEBNext® RNA Sample Purification Beads (supplied in NEB #E7755), rRNA depleted samples can alternatively be purified by ethanol precipitation or using RNA column cleanups such as the Monarch Spin RNA Cleanup Kit (NEB #T2030), For use with other columns, check the column specification for RNA size range to ensure any fragmented RNA will be retained during the cleanup. Q: Can the enriched RNA resulting from the NEBNext depletion kit be used in long read sequencing applications? A: Yes, but note that RNA is being somewhat fragmented during the depletion process. This can reduce read length. Q: Can I use NEBNext UltraExpress® RNA Library Prep Kit with NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) or NEBNext® RNA Depletion Core Reagent Set? A: It is possible to use NEBNext UltraExpress RNA Library Prep Kit with NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) or NEBNext RNA Depletion Core Reagent Set, however there is no official protocol. Please contact NEB Technical Support via info@neb.com to obtain instructions.