Large-scale siRNA screens can be used to identify genes that are directly or indirectly involved in a mechanism or pathway of interest. Typically the readout from these screens employs cell-based or reporter gene assays. A common step after siRNA screening is to validate the results from the screen with an independent method such as real-time RT-PCR to detect siRNA-induced knockdown of mRNA targets. Depending on the number of siRNAs tested, this validation can take longer than the screen itself. Two Applied Biosystems scientists have streamlined this step more than 10-fold, performing real-time RT-PCR on cells treated with over 1500 siRNAs targeting more than 500 genes within a single week. This streamlined workflow is discussed here.

Streamlined Workflow Reduces Experimental Time to a Single Week

For large-scale siRNA screens, the validation process can be very time-consuming. This article describes a streamlined workflow for measuring siRNA-induced knockdown of mRNA targets using real-time RT-PCR. By eliminating the laborious RNA isolation step and reducing the number of plates and tips needed to process the samples, this streamlined workflow enabled the analysis of target knockdown by over 1500 siRNAs in a single week with minimal hands-on time.

The workflow employs the TaqMan® Gene Expression Cells-to-CT™ Kit and TaqMan Gene Expression Assays to measure the effect of each siRNA on target gene expression. The speed of the Cells-to-CT methodology and robustness of the TaqMan real-time PCR approach maximize the throughput of the workflow while assuring reliable results (Figure 1). Two Applied Biosystems scientists demonstrated this workflow using Ambion® Silencer® Select siRNAs, which have been shown in side-by-side tests to yield better knockdown and more consistent data than competing technologies [1]. Silencer Select siRNAs also incorporate novel chemical modifications that reduce off-target effects by up to 90%.

Figure 1. High-Throughput siRNA Screening Workflow. This workflow is based on thirty-six 96-well plates. The time required for real-time PCR can be shortened further if more than one Applied Biosystems 7900HT Fast Real-Time PCR System is available.

siRNA Transfection

Silencer Select siRNAs were diluted and distributed into 96-well transfection plates using the Tecan Freedom EVO® 200 liquid handling system. The siRNA plate map was arranged so that each biological replicate was in a different area of the plate to reduce the impact of any positional effects on the data. The siRNAs were then reverse transfected into HeLa cells (4,000 cells per well) using Lipofectamine™ 2000 reagent (Invitrogen).

Positive and negative controls in siRNA experiments are critical, especially when processing large numbers of samples. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is widely recognized as a good target for positive control siRNAs because of its high, ubiquitous expression in virtually all mammalian cells. Positive controls for transfection efficiency, included Silencer Select GAPDH siRNAs and 3 in-house custom-designed siRNAs targeting ALDH1B1. Negative controls included untransfected cells and purified RNA, which were tested for GAPDH expression.

Sample Preparation

Samples were prepared for real-time PCR at 48 hours post-transfection, using the TaqMan Gene Expression Cells-to-CT Kit. This method was chosen due to its speed and ease of automation. Rather than isolating RNA prior to reverse transcription—a multiple step procedure that can take up to 60 minutes—the TaqMan Gene Expression Cells-to-CT Kit utilizes a novel buffer to lyse cells in 7 minutes at room temperature. The lysates can then be used directly as substrates for reverse transcription (RT) reactions. A CyBio CyBi®-Well vario liquid handler was used to pipette the Cells-to-CT reagents into the plates.

Real-Time RT-PCR

After sample preparation, reverse transcription was performed on the Applied Biosystems GeneAmp® PCR System 9700 with a 20% lysate input. In order to increase throughput and reduce the overall duration of the verification experiment, additional plates were prepared for cDNA synthesis using the Cells-to-CT procedure while other plates were undergoing their 60 minute RT incubation. Once the RT reactions were complete, the cDNA was transferred into custom 384-well plates that had been pre-loaded with the appropriate TaqMan Gene Expression Assays (see sidebar, Solutions for Streamlined Validation of siRNA-Induced Knockdown). Using pre-plated assays was another time saver. Finally, Gene Expression Master Mix and nuclease-free water were added to each well and the plates were run on a 7900HT Fast Real-Time PCR System using the robotic twister arm. For data analysis, the researchers used the ΔΔCT method to calculate relative knockdown. Knockdown was expressed in relation to samples treated with Silencer Select Negative Control #2 siRNA, using 18S rRNA as the normalizing gene. Data analysis procedures were similar to those discussed previously [2].

Faster Workflow Without Compromising Data Quality

By combining the TaqMan Gene Expression Cells-to-CT Kit, TaqMan Gene Expression Assays, and simple automation procedures into a single workflow, AB scientists were able to increase their weekly throughput for testing siRNA-induced knockdown more than 10-fold. A small subset of the results from a single week’s worth of work is shown in Figure 2. As expected, transfection with Silencer Select siRNAs resulted in high levels of target mRNA knockdown (Figure 2) with excellent reproducibility among transfections (Figure 3). Indeed, previous experiments [3] have demonstrated that reproducibility is not compromised by moving from an RNA isolation based methodology to the Cells-to-CT methodology, where reverse transcription is performed directly from the cell lysate.


Figure 2. Silencer® Select siRNAs Tested Using the Streamlined Workflow Showed Excellent Knockdown Efficiency. For each gene target (x-axis, lower labels), 3 siRNAs were transfected (x-axis, upper labels) with 4 replicates. Cells were lysed using the TaqMan® Gene Expression Cells-to-CT™ Kit, and real-time RT-PCR was performed directly in the cell lysates using the indicated TaqMan Gene Expression Assays. Knockdown data are expressed relative to data from cells transfected with Silencer Select Negative Control #2 siRNA. The positive controls are in-house custom siRNAs targeted to ALDH1B1.



Figure 3. High Replicate Reproducibility with the Streamlined siRNA Testing Workflow. For each siRNA, 4 replicate transfections were performed. Cells were lysed using the TaqMan® Gene Expression Cells-to-CT™ Kit, and real-time RT-PCR was performed directly in the cell lysates. Knockdown data were calculated relative to data from cells transfected with Silencer® Select Negative Control #2 siRNA. Variability was calculated by subtracting each replicate’s value from the median of the 4 replicates. The variability in target knockdown for each replicate, across all siRNAs transfected, is represented as box plots (see sideabar, How to Interpret a Box Plot).

Conclusion

This study demonstrates that large-scale siRNA screening and subsequent RT-PCR based knockdown validation can be made faster and easier by combining Silencer Select siRNAs, the TaqMan Gene Expression Cells-to-CT Kit, and pre-plated TaqMan Gene Expression Assays into a single workflow. Data quality is not compromised with the faster procedure, as demonstrated by excellent knockdown and high reproducibility.

Scientific Contributors
Rajeev Varma, Josquin Holmes, Angie Cheng, Susan Magdaleno • Applied Biosystems