The ultimate goal of most RNAi experiments is to understand the biological impact of the reduced target gene expression. Assays that measure the effects of gene silencing are varied and diverse. Morphological, enzymatic, biochemical, and immunological assays can all be useful depending on the goals of the experiment. In general, it is best to choose the simplest, most reproducible assay that is relevant to the biological process you are studying. However, first it is important to define the time course over which silencing takes place so that this window can be targeted in the biological assays. Because siRNAs exert their effects at the mRNA level, but phenotypes are usually induced by protein reduction, it is highly recommended that siRNA-induced silencing be measured at both the mRNA and protein levels. This is typically accomplished by qRT-PCR and Western blotting, respectively.

Silencing Time Course Methods
The window of mRNA and protein silencing was defined by delivering survivin and nontargeting siRNAs to cells and measuring mRNA (Figure 4) and protein levels (Figure 5) at 24, 48, 72, 96, and 120 hours post transfection. mRNA levels were monitored using qRT-PCR and the same TaqMan® Gene Expression Assay as used for verifying siRNA induced silencing in Step 3. To analyze survivin protein levels, Western blot analysis was performed on cell lysates using the Western-Light™ Immunodetection System. The Western-Light System provides a sensitive, chemiluminescent based protein detection method.

Methods for Measuring Apoptosis
A series of biochemical and morphological assays were performed to measure the effect of siRNA-mediated silencing of survivin on several apoptotic indicators over the time course described above.

Cell Survival
Relative cell survival was measured using fluorescein diacetate (FDA), a fluorogenic nonspecific esterase substrate.

Nuclear Condensation
Chromatin condensation is a late apoptosis indicator and was monitored by fluorescence microscopy after DAPI staining of the cells.

Increased phosphatidyl serine externalization
Induction of membrane asymmetry, as evidenced by phosphatidyl serine externalization, is an early apoptosis indicator and was measured by labeling with fluorescent annexin V and then assaying with an 8200 Cellular Detection System (Applied Biosystems).

Pro-caspase-3 activation
Caspase-3 activation is an early to mid-stage apoptosis indicator and was assayed using a fluorogenic caspase-3 substrate.

Silencing Time Course
Transfection of survivin siRNA into HeLa cells reduced survivin mRNA levels >80% compared to negative control transfected cells. However, the patterns of silencing were not identical. siRNA #2646 knocked down survivin mRNA over 80% consistently over the time course while survivin siRNA #121294 knocked down survivin mRNA 80% at 48 hours, but less efficiently at the later time points (Figure 4). The survivin protein expression pattern paralleled that of survivin mRNA expression (Figure 5).

Figure 4. Time Course of Survivin mRNA Reduction in HeLa cells Transfected with Survivin siRNAs. Silencer® siRNAs (#2646 and #121294) were transfected at 30 nM in triplicate into HeLa cells using 0.3 µL siPORT™ NeoFX™ Transfection Agent in 96 well plates (4000 cells/well). Total RNA was isolated from each sample at the indicated time point using the MagMAX™-96 Total RNA Isolation Kit. Survivin mRNA levels were measured using Survivin Hs00977611-g1TaqMan® Gene Expression Assay. Percent gene expression remaining was expressed as the relative amount of survivin mRNA in cultures transfected with survivin siRNA versus cells transfected with Silencer Negative Control #1 siRNA.

Figure 5. Time Course of Survivin Protein Reduction in HeLa Cells Transfected with Survivin siRNAs. Silencer® siRNAs (#2646 and #121294) were transfected in triplicate (30 nM siRNA) into HeLa cells using 5 µL siPORT™ NeoFX™ Transfection Agent in 6 well plates (2.5x105 cells/well). Survivin protein was detected in cell lysates harvested at the indicated time points by immunoblot using a rabbit polyclonal antibody to survivin (1:2000 dilution; Abcam Cat #AB469) and the Western-Light™ Immunodetection System (Applied Biosystems Cat #T1047). GAPDH was detected for use as a loading control (Ambion Cat. #AM4300). NT=Nontransfected; NC=Silencer Negative Control #1 siRNA.

Cell Survival
Survivin supports cell proliferation and thus, silencing of survivin might be expected to result in a decrease in cell number. This was tested and results are shown in Figure 6. As compared to negative control siRNA transfected cells, the two survivin siRNAs did not lead to significantly decreased cell numbers at any of the time points monitored.

Figure 6. Effect of Survivin Silencing by on Cell Number. Silencer® siRNAs #2646 and #121294 were transfected in triplicate (30 nM siRNA) into HeLa cells (4000 cells/well in 96 well plates) using 0.3 µL siPORT™ NeoFX™ Transfection Agent. At various time points post transfection, cells were harvested and lysed. Cell extract (8 µL) was added to 384 well plate wells containing 32 µL (0.01 mg/mL) fluorescein diacetate solution. Relative cell number/well was determined by measuring the increase in fluorescence (ex=488 nm, em=529 nm) over 4 min at room temperature.

Nuclear Condensation
Specific silencing of survivin has been shown to cause apoptotic events, one of which is characteristic changes in nuclear morphology due to nuclear condensation. Nuclear morphology was therefore observed 48–72 hours post transfection by staining cells with DAPI. At 48 hours, many survivin siRNA transfected cells exhibited nuclear condensation, while Silencer® Negative Control #1 siRNA transfected cells did not (Figure 7).

Figure 7. Survivin Silencing Causes Changes in Nuclear Morphology. HeLa cells transfected with (A) survivin siRNA #2646 (30 nM) and (B) Silencer® Negative Control #1 siRNA (Ambion, Cat. #AM4611), were fixed 48 hr post transfection, and stained with DAPI. Nuclear morphology was assessed using an Olympus BX60 fluorescent microscope.

Phosphatidyl Serine Externalization
One of the earliest hallmarks of apoptosis is phosphatidyl serine externalization. To see whether survivin silencing would cause an increase in phosphatidyl serine externalization, this parameter was measured by labeling with fluorescent Annexin V. HeLa cells were transfected with either the survivin siRNAs or negative control siRNA. 72 hours after transfection, fluorescent annexin V labeling was assayed using the 8200 Cellular Detection System (Figure 8). The 8200 instrument enables mix-and-read assays with live cells and beads. As with cell number, the observed magnitude of the effect varied somewhat between the two siRNAs.

Figure 8. Phosphatidyl Serine Externalization Caused by Silencing of Survivin. (A) HeLa cells were transfected at 4000 cells/well with 30 nM Silencer® survivin siRNA #2646, #121294, or Silencer Negative Control #1 siRNA (Ambion, Cat #AM4611) in 6 replicates using siPORT™ NeoFX™ Transfection Agent (0.3 µL). Cells were harvested at five time points (24, 48, 72, 96, and 120 hr; 72 hr time point shown) post transfection and assayed for phosphatidyl serine externalization using fluorescent labeled Annexin V (blue) and CentriRed™ DNA binding dye (pink) to enumerate cells. Cells were analyzed on the Applied Biosystems 8200 Cellular Detection System. (B) Cells transfected with negative control siRNA and Silencer siRNA #2646.

Caspase-3 Activation
A series of caspases are typically activated in the early stages of apoptosis. These proteases cleave key structural and nuclear proteins, which leads to chomosomal cleavage and nuclear condensation. Caspase-3 is generally the last caspase activated in the caspase cascade. Therefore we monitored the impact of survivin knockdown on activated caspase-3 levels. siRNA-mediated survivin silencing caused little or no effect on caspase-3 activity (data not shown). Our observation agrees with published reports stating that inhibition or silencing of survivin results in the activation of apoptosis events but does not activate caspases [1,2].

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References
1. Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, Chung CW, Jung YK, Oh BH (2001) An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochem 40: 1117–1123.

2. O’Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC, Altieri DC (2000) Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. PNAS USA 97: 13103–13107.