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Biowire Winter 2011 — Knockout Rats — More Effective Pre-Clinical Models

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Tech Tips: MISSION esiRNA for for RNAi Screening in Mammalian Cells

MISSION esiRNA (endoribonuclease-prepared siRNA) provides RNAi researchers with a proven, cost-effective, and simple way to perform RNAi screens.

Recently, the developers of esiRNA at the Max Planck Institute of Molecular Cell Biology and Genetics published a comprehensive video tutorial of an RNAi screen using MISSION esiRNA in the Journal of Visualized Experiments (JoVE)1. Below is an excerpt from the article. 

In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

High-Quality MISSION esiRNA Libraries

  1. For every gene of interest, the most susceptible and specific target region for RNAi is chosen utilizing the Design and Quality Control of RNAi (DEQOR) algorithm (http://nar.oxfordjournals.org/content/32/suppl_2/W113.full)2. DEQOR scores the silencing potential of all possible 21 nucleotide’s long siRNAs in a target-mRNA based on state-of-the-art design constraints2. In addition, each potential siRNA is analyzed for possible crossreactivity with other genes by performing a Basic Local Alignment Search Tool (BLAST) search against the transcriptome of the organism studied3. The program thereby provides an analysis of the overall quality and cross-silencing capacities of the potential esiRNA (300- 600 bp length).
  2. The chosen region of the target-gene cDNA is amplified by PCR using gene-specific primers flanked by bacteriophage RNA-polymerasepromoter sequences.
  3. Every PCR product used for MISSION esiRNA production is verified for identity by sequencing and for purity by Caliper LabChip® analysis.
  4. Long double-stranded RNA is transcribed from the PCR-product by RNA polymerase, followed by an annealing of the transcribed strands.
  5. esiRNAs are prepared by limited enzymatic digestion of the long double-stranded RNA using RNase III followed by purification via anion-exchange chromatography utilizing Q Sepharose® spin columns.
  6. esiRNAs are precipitated and resuspended in TE buffer. The yield is measured by UV-absorption measurements, and the concentration is adjusted by dissolving in an appropriate volume of TE buffer. The overall quality of the final product is checked by Caliper LabChip analysis.

Choosing a Cell Line and Optimizing Transfection for Screening

  1. Titrate amounts of esiRNA (use Eg5 as positive and Renilla luciferase (RLUC) as negative control) and increasing amounts of transfection reagent in a 384-well plate, add cells, and incubate for 48 hours. Eg5 is a kinesin motor protein required for bipolar spindle assembly, and depletion leads to a mitotic arrest. Repeat this procedure for different transfection reagents and cell lines. Here, we use HeLa cells cultured following standard procedures and Oligofectamine™ (Invitrogen) as transfection reagent.
  2. For choosing the optimal transfection conditions, count the cells transfected with Eg5 esiRNAs that show a mitotic arrest (round shape) and the total number of cells transfected with RLUC esiRNA by light microscopy. Choose the condition with the least toxicity for RLUC and the most pronounced phenotype for Eg5.

An alternative method for optimizing transfection conditions involves a stable cell line expressing EGFP (or another suitable reporter gene) under the control of a constitutive active promoter. After transfection of an esiRNA against EGFP (or another reporter gene) and RLUC (or another suitable negative control esiRNA) measure knock-down efficacy and toxicity (e.g., by fluorescence-assisted cell sorting). Make sure that the used esiRNA for EGFP is fully complementary to the target-mRNA.

Visit jove.com/index/details.stp?id=2008 to see the complete RNAi screening protocol including: Setting Up the Primary Screen, Primary Screen, and Secondary Screen and Hit Validation.

High false positive and false negative rates are a common challenge in RNAi screens. To address this problem, considerable efforts have been invested to improve the efficacy and in particular the specificity of the silencing triggers. An important discovery was that a pool of different siRNAs targeting the same transcript greatly enhances target specificity. Because a very complex pool of different siRNA s is produced by the endoribonuclease, esiRNA s are high target specificity triggers, reducing the false positive rate in RNA i screens. esiRNA s have also demonstrated to achieve efficient knockdowns, reducing also the false negative rate.

References

  1. Theis, M.; Buchholz, F. (2010). MISSION esiRNA for RNAi Screening in Mammalian Cells. JoVE. 39. www.jove.com/ index/details.stp?id=2008, doi: 10.3791/2008.
  2. Henschel, A.; Buchholz, F.; Habermann, B. DEQOR: a webbased tool for the design and quality control of siRNAs. Nucleic Acids Res. 32, W113-20 (2004).
  3. Altschul S.F.; Gish, W.; Miller, W. et al. Basic local alignment search tool. J. Mol. Biol. 215, 403-410 (1990).

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