RNA from tissues.

Friday, October 17, 2003

Description
Procedure for extraction of RNA from tissues.

Procedure
1. Place RNAlater stabilized tissue is to be used, in DEPC treated glass petridish for cutting,
and cut it. Place it into a suitably sized vessel for homogenization.

2. Disrupt tissue using homogenizer and homogenize tissue in Buffer RLT.
Note: Incomplete homogenization will lead to significantly reduced yields and can
cause clogging of the RNeasy column.

3.Centrifuge the tissue lysate for 10 min at 30005000 x g. Carefully transfer the supernatant to
a new 15 ml by pipetting. Use only this supernatant (lysate) in subsequent steps.
Note: In most preparations a small pellet will form, sometimes accompanied by a fatty
upper layer. Transferring the pellet or the fatty layer may reduce the amount of RNA
that binds to the membrane and cause the spin column to clog. To avoid transferring contaminants, hold the pipette tip underneath the fatty upper layer, and do not disturb the pellet.

4. Add 1 volume of 70% ethanol to the homogenized lysate, and mix immediately by shaking vigorously. Ensure that any precipitates are resuspended. Do not centrifuge. Continue without delay with step 5.
If some lysate is lost during steps 4 and 5, adjust volume of ethanol accordingly.
Note: Visible precipitates may form after the addition of ethanol when preparing
RNA from certain tissues (thymus, spleen, etc.). Resuspend precipitates completely
by vigorous shaking, and proceed immediately to step 5. Insufficient resuspension
of precipitates will cause DNA contamination and can lead to impure total RNA.

Mal Tissues
5. Apply the sample to a RNeasy midi column placed in a 15 ml centrifuge tube, and close the
tube gently. Maximum loading volume is 4.0 ml. Centrifuge for 10 minutes at 4000 rpm.
Discard the flow-through. Reuse the centrifuge tube in used in step 4.
If the maximum amount of starting material is used, it may be necessary to increase
centrifugation time in order to allow the lysate to completely pass through
the column.
If the volume exceeds 4.0 ml, load aliquots successively onto the RNeasy
column, and centrifuge as above. Discard the flow-through after each centrifugation
step

6. Add 4.0 ml Buffer RW1 to the RNeasy column. Close the centrifuge tube gently, and
centrifuge for 5 min at 30005000-x g to wash the column. Discard the flow-through.
Reuse the centrifuge tube from step 5.

7. Add 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube
gently, and centrifuge for 5 minutes at 4000 rpm to wash the column. Discard the
flow-through.
Reuse the centrifuge tube from step 6. In the RNeasy midi procedure, the flow-through
need not be discarded.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer
RPE before use.
Protocol
Tissues
8. Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, and
centrifuge for 5 min at 4000 rpm to dry the RNeasy silica-gel membrane.
It is important to dry the RNeasy membrane since residual ethanol may interfere with
downstream reactions. This centrifugation ensures that no ethanol is carried over during
elution.
Note: Following the centrifugation, remove the RNeasy column from the centrifuge
tube carefully so the column does not contact the flow-through as this will result in
carryover of ethanol.

9. To elute, transfer the RNeasy column to a new 15 ml collection tube (supplied with the kit).
Pipette the appropriate volume of RNase-free water directly onto the RNeasy silica-gel
membrane. Close the tube gently. Let it stand for 1 min, and then centrifuge for 5 min at
4000 rpm.

10. Repeat the elution step (step 9) as described with a second volume of RNase-free
water. To obtain a higher total RNA concentration, this second elution step may be
performed using the first eluate (from step 9). The yield will be 1530% less than the yield
obtained using a second volume of RNase-free water, but the final concentration will be
higher.

11. Eluted RNA is measured by spectrophotometer at 260 and 280 nm.

Recipes
1. Cups and rods for homogenization
2. Micropipettes and disposable pipette tips
3. Tissues stored in stabilizing liquid (RNAlater by Qiagen)
4. Qiagen RNeasy midi kit
5. 15 ml conical centrifuge tubes
6. 1.5 ml microcentrifuge tubes
7. Glass petridishes
8. Forceps
9. Scissors
10. NAOH/EDTA solution
11. DEPC treated water
12. 70% Ethanol (prepared in DEPC water)
13. 3M Sodium acetate (for precipitation)

Qiagen Reagent
Buffer RLT- for homogenization
Buffer RW1- wash buffer
Buffer RPE- wash buffer
Rnase-free water-for elution of RNA

Supplies

Tips

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RNA from tissues.