|
RNAi: Get the Whole Story
Mike Byrom, Ambion, Inc.
More and more researchers
are conducting siRNA experiments to elucidate cellular pathways
and gene function. Often the siRNA effect is only evaluated at
the level of mRNA, but this approach overlooks the true subject
of most siRNA studies--the biological effect caused by a reduction
in the target protein. The biological manifestations of knockdown
may not correlate directly with measured mRNA levels. Many proteins
remain functionally active long after the corresponding mRNA
has been removed. Other proteins need only be present in very
small quantities to have a profound biological effect. In these
situations, even a significant reduction at the mRNA level may
not be effective at producing a cellular response. To fully understand
the results of siRNA experiments, analyzing the knockdown of
both the target mRNA and the corresponding protein is recommended.
Isolate RNA and Protein from the Same Sample
Until recently, separate samples have
typically been required for isolation of high quality RNA and
protein. However, when working with rare or difficult-to-obtain
samples, it is sometimes impractical to isolate RNA and protein
independently. In studies involving large numbers of samples,
expensive reagents, or inherent variability (e.g. cell transfection),
using separate experimental samples to obtain RNA and protein
is not only costly and time consuming, but may also lead to
inconsistent results. Ambion's PARIS™ Kit was
developed specifically to address these problems. It allows
researchers to isolate both RNA and protein from a single experimental
sample. Figure 1 shows both Northern and Western data from
an siRNA experiment in which cells were transfected with either
a chemically synthesized siRNA or with a plasmid expressing
a hairpin siRNA--both targeting human GAPDH. The PARIS Kit
was used to isolate RNA and protein from either total cell
lysates, or from the nuclear or cytoplasmic cell fraction.
The cellular localization of GAPDH in the cytoplasm is reflected
in this data, which shows detection of GAPDH mRNA and protein
only in the cytoplasmic cell fraction.
|
|
|
Figure 1. Effects
of GAPDH siRNA on GAPDH mRNA and Protein Levels. HeLa
cells were plated at 200,000 cells/well into a 6 well
culture plate. 24 hours later they were transfected
with either a chemically synthesized siRNA at a final
concentration of 100 nM or with pSilencer™ 2.0-GAPDH;
both target human GAPDH. Samples were harvested 48
hours after transfection, and both RNA and protein
were isolated using the PARIS™ Kit. mRNA
knockdown was evaluated by Northern blot using an antisense
radiolabeled RNA probe transcribed from pTRI-GAPDH
human (Ambion Cat #7430). For the Western blot, anti-GAPDH
antibody (Ambion Cat #4300) was used.
|
Figure 2 also presents evidence at both the
mRNA and protein level for knockdown of two different genes,
Stat-1 and p53. In these experiments, cells were harvested for
analysis 72 hours after transfection. These results are typical
in that a significant RNAi effect can be seen 72 hours post-transfection.
|
|
|
Figure 2. Effects
of Stat-1 and p53 siRNAs on Target mRNA and Protein
Levels. HeLa cells
and 293T cells were plated at 30,000 cells/well into
24 well culture plates. The cells were transfected
using siPORT™ Lipid with an
siRNA targeting p53 (293T) or Stat-1 (HeLa) or with
a scrambled negative control siRNA (Silencer™ Negative
Control #1) at a final concentration of 100 nM. Samples
were harvested 72 hours after transfection and were
subjected to RNA and protein isolation using the PARIS™ Kit.
RNA was reverse transcribed using the RETROscript® Kit,
and target cDNA levels were analyzed by real-time PCR
using SYBR® Green detection. Target
gene expression in the transfected cells was compared
to cells transfected with an equal concentration of
the Silencer Negative Control
#1 siRNA. Input cDNA in the different samples was normalized
using real-time PCR data for 18S rRNA. The bar graphs
represent an average of three data points.
|
Timecourses: The Best Way to Understand
siRNA Effects
To get the best picture of the biological
effects of an siRNA on its target, we recommend conducting
a time course experiment to monitor both RNA and protein levels
over time. In Figure 3 a time course experiment was performed
after the introduction of an siRNA against PCNA (Proliferating
Cell Nuclear Antigen). Total RNA and protein were isolated
from the same sample at days 3, 4, and 5 and analyzed by either
real-time RT-PCR or Western blot. The maximal reduction of
message for PCNA was achieved by day 3; however, the maximal
reduction of protein did not occur until day 4. These data
emphasize the utility of the PARIS Kit for
analysis of both RNA and protein from your RNAi experiments.
Knowing when protein production is reduced will provide information
about when cellular phenotypes may arise.
|
|
|
Figure 3. Timecourse
of PCNA siRNA Effects on PCNA mRNA and Protein Levels. HeLa
cells were plated at 30,000 cells/well into a 24 well
culture plate. The cells were transfected with an siRNA
against PCNA or with the Silencer™ Negative
Control #1 (scrambled) at a final concentration of
100 nM using siPORT™ Lipid.
Duplicate samples were harvested at 72 hours and each
day for 5 days. The PARIS™ Kit was
used to isolate both RNA and protein for either real-time
RT-PCR (Panel A) or Western blot analysis (Panel B).
RNA was reverse transcribed using the RETROscript® Kit;
and target cDNA levels were measured by real-time PCR
using SYBR® Green assays. Input cDNA
in the different samples was normalized using real-time
data for 18S rRNA. The bar graphs represent an average
of three data points. Data are shown relative to mRNA
levels of cells transfected with scrambled control
siRNA.
|
Tools to Assist You in Your RNAi Research
Ambion offers products for analysis
of both mRNA and protein from the same sample; here we demonstrate
the versatility of the PARIS Kit for this
application. Other Ambion products used in this study include Silencer™ Validated
siRNAs to Stat-1, p53, and PCNA; and Silencer™ GAPDH
control siRNAs for eliciting RNAi. Also used were the RETROscript® Kit for cDNA synthesis, and antibodies
to GAPDH for analysis of
GAPDH at the protein level. Ambion's comprehensive suite of
products for analysis of siRNA experiments is appropriate for
anyone conducting research involving RNAi.
SYBR Green is a registered trademark of Molecular
Probes.
back
to top
|
