Biology Reference
In-Depth Information
3
Methods
3.1 Sample
Collection and
Microdissection
( See Note 1 )
1. Place surgical tissues resection specimen immediately on ice,
and subsequently snap freeze and store at −80 °C.
2. Prepare 5
m frozen sections from tissue blocks, place briefl y
in RNase-free ethanol, stain with hematoxylin and eosin
(H&E) and let sections to be diagnosed by a pathologist.
3. Section serially tissue blocks containing the piece of interest or
cell types (10
μ
m sections). Stain the slides with H&E and
immediately store at −20 °C.
4. Microdissect manually tissue section under a microscope (e.g.,
BH2, Olympus) using a sterile injection needle (size
0.65 × 25 mm) (
see
Note 2
).
5. Place microdissected cells into a 500
μ
μ
L microfuge tube con-
taining 100
L of lysis solution from the RNAqueous
®
-Micro
Kit (
see
Note 3
). Make sure that the sample is completely
immersed in lysis solution.
μ
3.2 RNA Purifi cation
We recommend the RNAqueous-Micro
®
Kit for RNA isolation.
1. Following incubation of the lysate at 42 °C for 30 min, per-
form all the following steps according to the manual of the
manufacturer (
see
Note 4
).
2. Omit DNase step if amount of total RNA is limited.
3.3 Evaluation
of RNA Quality
and Quantity
Quantitative evaluation of the isolated RNA should be performed
using a spectrophotometer which can handle small sample volumes
(1
L) such as the NanoDrop spectrophotometer. The assessment of
RNA quality can be performed using a BioAnalyzer. This is an auto-
mated bio-analytical device using microfl uidics technology that pro-
vides electrophoretic separations of the sample and analyzes RNA
using the same principles as denaturing agarose gels but requires
only 1 ng of total RNA. Profi les generated on BioAnalyzer yield
RNA integrity and ribosomal ratios. The ratio of the two rRNA 18S
and 28S is expected to be 1.8 for good quality RNA. The BioAnalyzer
software generates also the RNA Integrity Number (RIN). The RIN
is developed to extract information about RNA sample integrity
directly from the electrophoretic trace of the sample. The assigned
RIN score can vary from 1 to 10 where 10 indicates maximum integ-
rity and quality of the RNA (
see
Note 5
) (Fig.
3
).
1. Determine RNA quantity using a UV spectrometer (such as
NanoDrop) (
see
Note 6
).
2. Use 1 ng per sample for the assessment of RNA integrity using
BioAnalyzer LabChip
®
.
μ
