Large-scale siRNA screens can be used to identify genes
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
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 . Silencer Select siRNAs also incorporate novel chemical modifications that reduce off-target effects by up to 90%.
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.
Faster Workflow Without Compromising Data Quality
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).
Rajeev Varma, Josquin Holmes, Angie Cheng, Susan Magdaleno • Applied Biosystems
The box extends from the lower quartile (25th percentile) to the upper quartile (75th percentile), representing the middle 50% of the data. The length of the box represents the inter-quartile range (IQR). The median is shown as a line within the box. The vertical lines extending outside the box, also called whiskers, represent data that lies within 1.5 X IQR of the quartiles. Data that fall outside the whiskers, referred to as outliers, are shown as circles.
With the TaqMan Gene Expression Cells-to-CT Kit, RNA purification from cultured cells is no longer necessary prior to real-time RT-PCR. The kit includes reagents to lyse cultured cells (with the option to simultaneously remove genomic DNA), optimized reverse transcription reagents to synthesize cDNA directly from the lysate, and TaqMan Gene Expression Master Mix for real-time PCR. Quantitate targets of interest using TaqMan Gene Expression Assays.
TaqMan® Gene Expression Assays
Applied Biosystems offers more than 1,000,000 TaqMan Gene Expression Assays for a broad assortment of species—the most comprehensive set of pre-designed real-time PCR assays available. All TaqMan Gene Expression Assays have been designed using our validated bioinformatics pipeline, and can be thermal cycled with the same PCR parameters, eliminating the need for primer design or PCR optimization.
TaqMan® Express Plates
New TaqMan Express Plates are a cost-effective and convenient 96-well format complementing our line of individual TaqMan Gene Expression Assays. Any of the 6 available plate formats can be customized with dried-down TaqMan Assays in specific well locations. This flexibility enables efficient screening of biological samples for the presence and amount of specified gene targets expressed in human, mouse, rat, canine, and rhesus species.
TaqMan® Custom Plating Service
TaqMan Custom Plating Service offers the convenience of pre-plated TaqMan Gene Expression Assays, Custom Assays, and Custom Probe/Primer Sets in 96- or 384-well plates. Set up custom configurations using TaqMan Gene Expression Assays (Inventoried, Made-to-Order, and Custom) and Custom TaqMan Probes and Primers, enabling analysis of genes from any organism. Select from a variety of reaction volumes, and receive the plates in dried or liquid formulation.
- Puri N, Wang X, Varma R, Latham K, Sendera T, Magdaleno S (2008) Next Generation siRNAs to Make Your Silencing Roar. TechNotes 15(1):7−10.
- Varma R, Cheng C (2008) Tips from the Bench: Calculating Gene Expression Changes and Variability in siRNA Experiments. TechNotes 15(2):10−11.
- Chapman L, Nguyen A, Fekete R, Stevens J, Dong S, Liu C (2007) Real-Time RT-PCR Directly from Cell Lysates: A Complete Workflow without RNA Purification. TechNotes 14(3):23–25.
- Tukey, J.W. “Box-and-Whisker Plots.” Exploratory Data Analysis. Reading, MA: Addison-Wesley, pp. 39-43, 1977.