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he same experimental sample (Figure 1).
Figure 1. Schematic of PARIS Procedure.
Using PARIS, RNA and protein can be isolated simultaneously from whole
cell lysates. Alternatively, RNA and protein can be isolated from separate
nuclear and cytoplasmic fractions (see "Application: Nuclear vs.
Cytoplasmic Fractionation" at right). Tissue or cultured cells are
first homogenized in ice-cold Cell Disruption Buffer to prepare a total
cell lysate. Since the homogenization is performed quickly on ice and
in the presence of detergent, both protein and RNA can be purified directly
from this lysate. For RNA isolation, a part of the total cell lysate is
immediately mixed with an equal volume of Lysis/Binding Solution. This
solution contains a high concentration of guanidinium thiocyanate, a strong
chaotropic denaturant that rapidly inactivates cellular ribonucleases.
Total RNA is then purified from the mixture using an RNA binding glass
fiber filter. After three rapid washing steps, high quality RNA is eluted
in a concentrated form. The entire procedure can be completed in less
than 20 minutes. Note: This kit is not recommended for tissues with high
levels of ribonucleases, such as pancreas.
Compatible with Most Downstream Applications
The RNA isolated from total, nuclear, or cytoplasmic fractions with the
PARIS procedure can be used in a variety of downstream applications, including
blot hybridization, in vitro translation, cDNA synthesis, and RT-PCR.
A DNase I treatment is recommended for RNA that will be used for RT-PCR
experiments, especially if using primers that do not flank introns, or
for genes that have processed pseudogenes. Ambion's
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