Optimizing the workflow for successful RNA interference (RNAi) experiments in difficult-to-transfect cells



A well-planned gene silencing experiment – particularly when using cell lines that are difficult to transfect – requires optimal delivery of functional silencing reagents into the cell and accurate confirmation of gene knockdown. Horizon offers a proven, step-by-step experimental workflow, to ensure confidence in the gene silencing methodology.

RNAi silencing is an elegant tool for characterizing a specific phenotype or disease state. Based on the endogenous biological pathway, it can be used for detailed study of a particular gene target or a defined biological pathway, or a high-throughput screen of an entire genome.

Successful RNAi experiments rely on four key steps:accell blog image of purple cells

  1. Obtain functional siRNAs
  2. Optimize delivery of siRNAs
  3. Detect gene down-regulation
  4. Investigate the biological phenotype

                                                                                                                

There are various barriers that can prevent one from achieving the desired experimental results with conventional methods. In particular, effective delivery of siRNA is often challenging, as many biologically relevant model systems used in immune and neurological studies depend on cell types that are refractory to conventional lipid delivery systems.

Horizon recommends that you use Accell siRNA technology to overcome some of these challenges and optimize the first two stages:

Accell siRNA workflow

Figure 1. Workflow for successful RNAi gene silencing experiments

This innovative technology provides functional and specific siRNAs that can be delivered without transfection reagents, viral vectors or instrumentation, and supports effective delivery to a wide range of ‘difficult-to-transfect’ human, rat and mouse cell lines.

Horizon offers a step-by-step experimental workflow to support optimizing RNAi silencing using Accell siRNA technology and reagents. After following these steps, you can confidently proceed to the study of the resulting biological phenotype through high content or endpoint assays.

Experimental workflow for Accell siRNA-mediated gene silencing and detection of mRNA knockdown

accell workflow 2Figure 2. Experimental workflow for Accell siRNA-mediated gene silencing and detection of knockdown.

For more detailed guidance on this workflow, see our Accell application note. The success of siRNA delivery and subsequent gene knockdown when following this workflow has been demonstrated in two difficult-to-transfect cell lines (SH-SY5Y [adherent neuroblastoma cells] and Jurkat [suspension T cells]). Here is a summary of the findings:

  • Effective uptake of dye-labeled siRNA into the cytoplasm of both cell types was observed via fluorescent microscopy 24 hours after delivery (the optimum time for detecting mRNA knockdown is usually 72 hours post-delivery).
  • Neither cell line was adversely affected by Accell application; viability was >95% compared to untreated cells.
  • Expression of two target genes (GAPD and PPIB) was reduced in SH-SY5Y cells by 95% and 75%, respectively and in Jurkat cells by 93% and 60%, respectively. In contrast, there was no significant effect on a non-targeting housekeeping gene.

By following this recommended workflow, your RNAi experiments should perform as expected, even in ‘difficult-to-transfect’ cell lines.

More detail on these validation data, as well as the specific methodology, can be found in our application note Successful RNA Interference Experiments in Difficult-to-Transfect Cell Lines.

 

 

Christian_Nievera_headshotChristian Nievera, Ph.D., Product Manager

Christian is the Senior Product Manager for RNAi and functional genomics screening libraries at Horizon Discovery. He has over a decade of experience in the life science industry managing varied portfolios including: antibodies, siRNA, gene editing tools, and PCR reagents. Christian collaborates with research teams to develop and bring novel functional genomics tools to market. Christian earned his Ph.D. in Biological Sciences from Florida Tech.

 

 

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