Protocol content (NEB DNAse I reaction Protocol) :
RNA: 10ug
DNAse reaction buffer 1X 10ul
DNAse (Rnase free) 1ul
Nuclease free water 100 ul
My question is; if I use this protocol after isolating total RNA, I dilute the RNA ratio.
If I use it during RNA isolation, I am not yet getting RNA according to the protocol.
At what stage should I use?
Thank you!
what do you mean exactly by diluting the ratio? and what kit did you use for extraction? If you already have your RNA then you carry out the DNAse digestion then the sample is reconcentrated using phenol-chloroform extraction. In certain RNA extraction kits (qiagen for example) the DNAse digestion is incoprorated into the protocol (although they use a propitiatory buffer to allow on-column digestion using their DNAse).
Hanna Alalam
I use the qiagen kit for Rna extraction. As you say, I should add the DNAse application to the qiagen protocol, but at what stage?
After obtaining the RNA, there is a DNAse protocol with a total volume of 100 ul as indicated below.
Wouldn't I be diluting the existing RNA when I implemented this protocol?
Because of this idea, I cannot be sure at what stage I should do DNAse application.
If you use post extraction digestion then you just dilute for the extraction protocol. However, since you will need to purify again after the digestion to remove the DNAse you will reconcentrate the sample to the original concentration. For example if you eluted with 30ul for the original extraction then you can digest in a total volume of 100ul (you diluted here). Then after the digestion you do phenol chloroform and afterwards resuspend in 30ul again therefore the only amounts that you lose is what is lost during the precipitation. I suggest is that you follow the on column DNAse digestion to circumvent going through all of this miss it is written in the kit's appendix D and I will copy it here for reference (just be sure to use the DNAse set that is from qiagen since on column digestion is only compatible with their set ) essentially you split the RW1 wash step into two first you wash with a smaller volume of RW1 then incubate the column with dnaseI suspended in buffer RDD. Then you wash away with the seconds half of RW1 buffer (to remove the dnase I)
Full protocol (copied from RNAeasy kit):
Appendix D: Optional On-Column DNase Digestion
with the RNase-Free DNase Set
The RNase-Free DNase Set (cat. no. 79254) provides efficient on-column digestion of
DNA during RNA purification. The DNase is efficiently removed in subsequent wash
steps.
Note: Standard DNase buffers are not compatible with on-column DNase digestion.
Use of other buffers may affect the binding of RNA to the RNeasy membrane, reducing
RNA yield and integrity.
Lysis and homogenization of the sample and binding of RNA to the RNeasy membrane
are performed according to the standard protocols. After washing with a reduced
volume of Buffer RW1, the RNA is treated with DNase I while bound to the RNeasy
membrane. The DNase I is removed by a second wash with Buffer RW1. Washing with
Buffer RPE and elution of RNA are then performed according to the standard protocols.
Important points before starting
Generally, DNase digestion is not required since RNeasy technology efficiently
removes most of the DNA without DNase treatment. However, further DNA
removal may be necessary for certain RNA applications that are sensitive to very
small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundant target).
DNA can also be removed by a DNase digestion following RNA purification.
Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physical
denaturation. Mixing should only be carried out by gently inverting the tube.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
† To make a saturated solution, add solid bromophenol blue to distilled water. Mix and continue to add more
bromophenol blue until no more will dissolve. Centrifuge to pellet the undissolved powder, and carefully
pipet the saturated supernatant.
RNeasy Mini Handbook 06/2012 67
Things to do before starting
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first
time. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNasefree
water provided. To avoid loss of DNase I, do not open the vial. Inject RNasefree
water into the vial using an RNase-free needle and syringe. Mix gently by
inverting the vial. Do not vortex.
For long-term storage of DNase I, remove the stock solution from the glass vial,
divide it into single-use aliquots, and store at –20°C for up to 9 months. Thawed
aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots
after thawing.
Procedure
Prepare and load samples onto the RNeasy spin column as indicated in the individual
protocols. Instead of performing the first wash step, follow steps D1–D4 below.
D1. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane.
Discard the flow-through.*
Reuse the collection tube in step D4.
D2. Add 10 µl DNase I stock solution (see above) to 70 µl Buffer RDD. Mix by gently
inverting the tube, and centrifuge briefly to collect residual liquid from the sides of
the tube.
Buffer RDD is supplied with the RNase-Free DNase Set.
Note: DNase I is especially sensitive to physical denaturation. Mixing should only
be carried out by gently inverting the tube. Do not vortex.
D3. Add the DNase I incubation mix (80 µl) directly to the RNeasy spin column
membrane, and place on the benchtop (20–30°C) for 15 min.
Note: Be sure to add the DNase I incubation mix directly to the RNeasy spin
column membrane. DNase digestion will be incomplete if part of the mix sticks to
the walls or the O-ring of the spin column.
D4. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.*
Continue with the first Buffer RPE wash step in the relevant protocol.
Note: In most of the protocols, the immediately following Buffer RW1 wash step is
skipped (as indicated in the protocol). Continue with the first Buffer RPE wash step.
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