Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 135

be tailored to the cell or tissue type under study. Whereas most
cultured cells can be homogenized by simply vortexing in a cell
lysis solution, animal or human tissues, require more rigorous
methods of disruption, such as Qiagens TissueRuptor or
TissueLyser.

This protocol has been adapted and optimized from the Qiagen
miRNeasy Mini Kit.

2.2.3. Procedures

1. Place the tissue into a suitable sized vessel for disruption and
homogenization and add 1 ml of Lysis Reagent (QIAZOL).
Be sure that the entire tissue surface is completely surrounded
by the buffer to quickly permeate the tissue.
2. Disrupt and homogenize the tissue with the preferred method.
3. Place the tube containing the homogenate on the benchtop at
room temperature for 5 min.
4. Add 1 vol (200 l) of chloroform to the tube containing the

homogenate and close it securely. Shake it vigorously for 15 s.
5. Place the tube on the benchtop at room temperature for 2-3 min.
6. Centrifuge for 30 min: 12,000× g at 4°C.
Notes: Place the tubes on ice, especially if extracting a large
number of samples. After centrifugation, change the tempera-
ture to 15-25°C if the same centrifuge will be used. After cen-
trifugation, the sample separates into three phases: an upper
colorless aqueous phase containing RNA, a white interphase,
and a lower red organic phase.
7. Transfer the upper aqueous phase to a new collection tube
(approx. 530

μ

l).
8. Add 1.5 vol (here 800

μ

l) of 100% ethanol and mix thoroughly
by pipetting up and down several times.
9. Immediately transfer up to 700

μ

l of the sample to an RNeasy
spin column (2 ml collection tube).
10. (a) Close the lid and centrifuge for 15 s at ³8,000× g
(³10,000 rpm). Discard fl ow-through.
(b) Repeat step 4 until the whole sample has passed through
the membrane. Discard the fl ow-through each time.
11. (a) Add 700

μ

l buffer RW1 to the RNeasy Spin column. Close
the lid and centrifuge for 15 s at ³8,000× g (³10,000 rpm) to
wash the spin column membrane. Discard the fl ow-through.
(b) Repeat this step.
12. (a) Add 500

μ

l buffer RPE to the RNeasy spin column. Close
the lid gently and centrifuge for 15 s at ³8,000× g
(³10,000 rpm) to wash the spin column membrane. Discard
the fl ow-through.
(b) Repeat this step.

μ