New England Biolabs, E7865L, NEBNext® RNA Depletion Core Reagent Set

Introducing a new solution for customized depletion of abundant RNAs. Related Categories RNA Depletion & mRNA Enrichment,, Epitranscriptome Analysis,, RNA Library Prep for Illumina, FAQ Q: How can I design DNA probes to use with the NEBNext RNA Depletion Core Reagent Set? A: You can design single stranded DNA probes using the NEBNext Custom RNA Depletion Design Tool. You will need to provide the RNA sequence you desire to deplete 5’ to 3’ in FASTA format. Q: Are the probes designed by the NEBNext Custom RNA Depletion Design Tool sense or antisense? A: The resulting probes will be antisense to the RNA sequence you input into the NEBNext Custom RNA Depletion Design Tool. Q: Can the input sequence into the NEBNext Custom RNA Depletion Design Tool contain IUPAC Ambiguity Code? A: No. The input sequence should only contain A, C, G or T nucleotides. Q: Does the NEBNext Custom RNA Depletion Design Tool check for probes targeting transcripts other than the ones provided? A: No. The NEBNext Custom RNA Depletion Design Tool designs probes based on the RNA sequence provided. When selecting the RNA sequence to deplete, it is recommended to check for sequence homology with other transcripts of interest. Q: I have used NEBNext Custom RNA Depletion Design Tool and obtained probe sequences, what’s the next step? A: The next step is to order the probes from your preferred oligo synthesis provider. Standard desalting oligo purification is sufficient for this application. No modifications at the 5′ or 3' ends are needed. Refer to the manual for the NEBNext RNA Depletion Core Reagent Set for further details. Q: How do I make a working probe pool to be used with the NEBNext RNA Depletion Core Reagent Set? A: We recommend preparing an equimolar probe pool where probes are between 2-4 µM each, then using 1-3 µl of this probe pool in the probe hybridization step of the NEBNext RNA Depletion Core Reagent Set protocol. Optimization of the amount of probe used might be required for efficient depletion of your desired RNA. Q: What’s the maximum number of probes that can be combined in a pool? A: The depletion protocol has been tested using up to 1,000 probes. The use of a larger probe pool will require testing. Q: Can I use this product with degraded RNA or fragmented RNA? A: Yes, this kit can be used with degraded or fragmented RNA. Q: What total RNA input should I use with the NEBNext RNA Depletion Core Reagent Set? A: We recommend using 10-1000 ng total RNA. 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: How can I determine if the RNA depletion was efficient? A: To assess depletion efficiency, design RT qPCR primers for the depleted RNA, and primers for a housekeeping gene. Compare the RNA content before and after depletion. Q: Can I use Agencourt®AMPure XP Magnetic Beads instead of RNAClean® XP Magnetic Beads/NEBNext® RNA Sample Purification Beads? A: Yes, it is possible to use AMPure XP Beads. However, we recommend using the RNAClean XP Beads/ NEBNext® RNA Sample Purification Beads as these beads have been manufactured and tested to eliminate RNase contamination. A version of the kit includes these beads: NEBNext® RNA Depletion Core Reagent Set with RNA Sample Purification Beads (NEB #E7870). Q: Can I use purification methods other than NEBNext® RNA Sample Purification Beads/Agencourt® RNAClean® XP Magnetic Beads with the NEBNext RNA Depletion Core Reagent Set? A: Although we recommend using RNAClean® XP beads NEBNext® RNA Sample Purification Beads (supplied in NEB #E7870), RNA-depleted samples can alternatively be purified by ethanol precipitation or using RNA column cleanups such as the Monarch 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: Does the NEBNext Custom RNA Depletion Tool check for probe redundancy or overlap between target RNA sequences entered? A: No. The NEBNext Custom RNA Depletion Tool designs probes to all entered sequences independently, even if entered at the same time. It does not check for probe redundancy/overlap between multiple entries. 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.