Genome-wide siRNA and microRNA High-throughput Screens

Genome-wide siRNA and microRNA High-throughput Screens

Altogen Labs provides genome-wide siRNA and miRNA (microRNA) high-throughput screens (HTS) for RNAi research. RNAi HTS technology enables researchers to simultaneously screen thousands of loss-of-function genes and identify the association of genes with their corresponding biological phenotypes. The powerful new technology of whole-genome RNAi HTS has produced enormous amounts of data that has accelerated biological research, gene function studies, and medical discovery.

At Altogen labs we focus on gene target discovery to identify potential targets for therapeutic treatment and compound inhibition as well as target novel proteins involved in disease pathways. Our scientists bring years of extensive cell culture experience and expertise in the research and development of novel drugs and companion diagnostic tests. While working with Altogen Labs you can be confident that comprehensive analysis of data collected during genome-wide HTS will be fully examined and validated to determine the most promising therapeutical targets.

Inducing gene silencing (RNAi) in cell lines is powerful research approach to study gene function. Altogen Labs provides a number of RNAi services: RNAi assay development, in vivo siRNA synthesis, encapsulation, and administration, siRNA transient and shRNA stable transfection, RNAi cell-based library screening, siRNA/shRNA design, synthesis, in vitro validation, qRT-PCR test of target mRNA knockdown, development of RNAi cell lines, mRNA reduction in tissues, siRNA targeting, chemical modification, cell transfection optimization, siRNA and microRNA genome-wide library screening.

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Screening siRNA libraries is a powerful method for identifying individual genes required for cell function (i.e. cell survival, proliferation, migration, differentiation, invasion) or for sensitivity/resistance to specific drug effects. siRNA libraries consist of arrays of custom designed siRNAs specifically targeting known gene transcripts. Commercially available libraries consist of arrays of individual  siRNAs or pools of 2-3 unique, non-overalapping  siRNAs targeting the same gene. Multiple formats are available to fulfill specific experimental needs, including genome-wide coverage (siRNAs targeting all known human or mouse mRNA transcripts), pathway specific libraries (siRNA pools target transcripts of genes known to be involved in specific functional pathways, such as EFGR signaling pathway, kinases, tumor suppressors, etc), custom siRNA libraries (custom arrays of pools can be defined for a clients specific needs).

Example of a procedure used to identify  human kinases required for breast cancer cell growth.  In this example a primary screen used a siRNA library containing a pool of unique siRNA pairs each targeting 639 individual kinase genes. A set of 28 kinases were identified and used for secondary screening.

Figure 1. Identification of kinases involved in breast cancer cell growth. A breast cancer cell line was transfected with a siRNA library that targeted a variety of specific kinases. Adapted from Hu et al. Cancer Research 2012, 14:R22.

siRNA library screening protocol:

1)  Transfection of cells with siRNA pools is performed using reverse transfection:

  • Prepare siRNA:liposome complexes in the wells of 96-well plates
  • Add cells to the wells and incubate for 24 hours to allow cells to take up the siRNAs and to attach to the plate
  • Controls:   Cells treated with lipid reagent alone without siRNA.  Cells transfected with scrambled siRNAs

2)  Change medium and incubate cells for 48 hours for cell growth.

3)  Using plate reader assay cell viability (optional: cell proliferation, apoptosis, cell cycle, etc).

4)  Data analysis:  Only those siRNAs that each sequence showed a minimum of 30% inhibition compared with control were used for the secondary screen

Important considerations when planning experiments:

Cell types to use – many cell types are available for immediate use from ATCC and DSMZ. In addition, client derived cell lines and custom engineered cell lines can be generated by Altogen Labs scientists.

Transfection conditions:  Successful screening requires optimal transfection conditions for each cell line.  If transfection conditions are not known for a specific cell line then Altogen Labs scientists will perform transfection optimization.

Validation of gene silencing (RNAi) is verified by real-time qRT-PCR or by high throughput quantitative Western Blot analysis.

Successful screening requires a robust and informative assay.  Altogen Labs offers a variety of cell based and biochemical high throughput assay systems.  These assays include the measurement of apoptosis, growth arrest, specific kinase activity and protein phosphorylation.  Screening assays used can be commercially available, client developed or custom assays developed by Altogen Labs scientists.

Once we know the details of your project, we can provide you an immediate price quote (contact e-mail: info@altogenlabs.com or call Altogen Labs technical support at 512-433-6177). Please note that experimental details will help us to provide an accurate quote and timeline estimate.

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