permits transition of previously frozen samples to a state compatible
with handling at room temperature and standard homogenization methods
(see the article"Stop
Grinding Frozen Tissue
" in this issue). As shown in Figure
3, the PARIS procedure is compatible with both of these tissue collection/RNA
stabilization reagents for both cells and tissue. After homogenization,
protein samples are readily available for applications that do not
require native functional protein, such as Western blot analysis.
Figure 2. 2D Analysis of Proteins Isolated Using the PARIS Kit.
Protein samples were resolved using a pH 4-7 IPG gel followed by 8-16%
SDS-PAGE. Top: Total protein (~25 g) from HeLa cells stained with
SYPRO Ruby. Bottom: Total protein (~125 g) from mouse brain
stained with Coomassie Blue.
Figure 3. RNA and Protein Isolation from Stabilized Samples. (A)
Isolation from ~30 mg of mouse liver, brain or kidney 5 days after collection.
Samples were either kept frozen at -80C, stored in RNAlater
at 4C, or snap frozen and then stored in RNAlater-ICE at -20C.
(B) Isolation from 106 HeLa cells freshly harvested, kept 2 days
in RNAlater at RT, or in RNAlater-ICE at -20C.
Figure 4. Analysis of the RNAi Effect. (A) 5 x 104
HeLa cells were transfected in a 24 well plate with the indicated plasmids.
48 hours after transfection, total RNA and protein were isolated with
the PARIS Kit and analyzed by denaturing agarose gel electrophoresis,
Northern blot, or Western blot. SCR represents a scramblPage: All 1 2 3 4 5 Related biology technology :1
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