FAQ: Can I use more than one type of NEBNext Depletion Solution in the NEBNext RNA depletion workflow?

Yes, it is possible to combine probes. Any combination of NEBNext Depletion Solutions (NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7400/E7405); NEBNext rRNA Depletion Kit  (Bacteria) (NEB# E7850/E7860); NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7750/E7755) can be used at the probe hybridization step.

  • The volume of the total sample during the probe hybridization step must remain constant (15 µl).
  • The volume of the probe hybridization buffer must always remain 2 µl.
  • The volume of the individual depletion solutions should be determined experimentally with an input titration experiment, since the optimal amount of probes will vary from sample type to sample type. In the absence of this experimental data, the probes can be added in a 1:1 ratio.
  • Since the total volume of the reaction is 15 µl, the volume of total RNA that can be added is dependent on how much volume is left after adding the 2 µl Hybridization Buffer and the volume of the Depletion Solutions. If you are diluting your total RNA in nuclease free water prior to depletion, the volume of nuclease free water can be reduced to accommodate a higher volume of NEBNext Depletion Solution.



Total RNA in Nuclease-free Water (10 ng–1 μg)

Variable, to 11 μl

Combination of Depletion Solutions


 (white) NEBNext Probe Hybridization Buffer

2 μl

Total Volume

15 μl



Please refer to the protocol in the NEB #E7400, NEB #E7750 or NEB #E7850 manual.