Biology Reference
In-Depth Information
Tissue Mini Kit in accordance with the manufacturer's protocol
(
see
Note 3
).
1. The tissue is transferred from the tube containing the
RNAlater solution to a fresh 2.0-ml Eppendorf tube con-
taining 1 ml of the QIAzol
®
lysis reagent and a stainless steel
bead (
see
Note 4
). The tissue is subjected to shredding at
50 Hz for 10 min at 15-25 °C using a TissueLyser LT
(QIAGEN) (
see
Note 5
).
2. The stainless steel bead is removed, and 200
l chloroform is
added to the tissue lysate. The mixture is vortexed thoroughly
and centrifuged at maximum speed for 15 min at 4 °C.
3. After centrifugation, the mixture of tissue lysate and chloro-
form has separated into three phases. The upper aqueous phase
contains the RNA, the interphase contains the DNA, and the
lower phase contains the chloroform and proteins. The upper
RNA-containing phase is transferred into a fresh 1.5-ml
Eppendorf tube, 600
μ
l isopropanol is added, and the mixture
is vortexed thoroughly for 1 min (
see
Note 6
).
4. A 700-
μ
l sample of the solution is transferred to an RNeasy
®
Mini Spin column and centrifuged at 10,000 rpm for 15 s. The
fl ow-through is discarded, and the RNA is bound to the quartz
membrane of the RNeasy column.
5.
Step 4
is repeated using the remainder of the RNA-containing
mixture.
6. To remove any membrane-bound chromosomal DNA, the
membrane is treated with DNAse I. The DNAse I solution is
prepared by mixing 70
μ
μ
l RDD (RNeasy
®
) buffer with 10
μ
l
l) is pipetted onto the middle of
the column, and the column is incubated for 19 min at
15-25 °C.
7. The column is washed with 700
DNase I. This solution (80
μ
l RW1 buffer (RNeasy
®
)
under the same centrifugation conditions as previously
described. The fl ow-through is discarded.
8. The column is washed twice with 500
μ
l RPE buffer contain-
ing ethanol (RNeasy
®
). The column is centrifuged using the
conditions described above.
9. The column is dried by centrifugation for 2 min at maximum
speed.
10. The column is transferred into a 1.5-ml Eppendorf tube.
11. To elute the total RNA from the column, 30
μ
μ
l RNase-free
water is pipetted onto the quartz membrane.
12. After incubation for 1 min at 15-25 °C, followed by centrifu-
gation for 1 min at 11,000 rpm, the eluted RNA solution is
frozen and stored at −80 °C.
