Biology Reference
In-Depth Information
8. The column is incubated 15 min at RT with 80
μ
L DNase
solution consisting in 10
μ
L of DNaseI and 70
μ
L of digestion
buffer. Then, 350
L of RW1 buffer are added to the column
and centrifuged 15 s at
μ
≥
8,000 ×
g
at RT. The fl ow-through is
discarded.
9. Add 500
μ
L of RPE buffer to the column and centrifuge 15 s
8,000 ×
g
at RT. The fl ow-through is discarded.
10. Add 500
at
≥
μ
L of RPE buffer to the column and centrifuge 2 min
8,000 ×
g
at RT. The fl ow-through is discarded.
11. The column is further centrifuged 1 min at RT to eliminate
contaminating ethanol and to dry the membrane.
12. To elute, the column is placed in a new 1.5 mL collection tube
and 50
at
≥
L of RNase-free water are pipetted directly to the
membrane. After incubation for 1 min, the column is centri-
fuged 1 min at
μ
8,000 ×
g
at RT.
13. RNA concentration is determined spectrophotometrically,
where 1 OD at 260 nm corresponds to 44
≥
g/mL, and the
quality of the RNA samples may be analyzed on a standard 1 %
agarose gel, 1× TAE gel.
14. RNA samples are stored at −80 °C (
see
Note 1
).
μ
1. Use Disposable RNA chips to determine the concentration
and purity/integrity of RNA samples using Agilent 2100
bioanalyzer, as described in the detailed protocol provided by
Agilent
3.6 RNA Analysis
and Quantifi cation
Technologies
(
http://www.home.agilent.com/
-
Agilent 2100 bioanalyzer).
2. The electropherogram of a sample for total RNA (eukaryotic)
should resemble the one shown on Fig.
1
.
3.7 Chip
Hybridization
and Scanning
1. cDNA synthesis, biotin-labeled target synthesis, GeneChip
arrays hybridization, staining and scanning are performed
according to the standard protocol and technical manual of
instrumentation supplied by Affymetrix (
http://www.affyme-
2. Affymetrix Command Console Software is used to perform all
technical steps of the procedure and to produce the fi nal data.
(*.CEL fi les).
3.8 Microarray
Data Analysis
1. Software packages can be used for data analysis. Probe level
data from *.CEL fi les can be obtained, normalized, and con-
verted to expression values using Partek Genomics Suite
(Partek, Inc.), following the RMA algorithm [
16
] or DChip
procedure (invariant set) [
17
].
