You need to find the best RNA interference tools for your biotech lab. What are your options?

1 siRNA delivery

siRNAs are short, synthetic, double-stranded RNA molecules that can bind to complementary mRNA sequences and trigger their degradation. They are the most common and versatile RNAi tools, but they also have some challenges. The main one is delivering them to the target cells, especially if they are in vivo or hard to transfect. You can use various methods, such as liposomes, nanoparticles, electroporation, or viral vectors, depending on your cell type, tissue, and budget. You should also optimize the siRNA design, concentration, and duration to achieve efficient and specific knockdown.

2 shRNA expression

shRNAs are short hairpin RNAs that can be expressed from a plasmid or a viral vector and processed into siRNAs by the cellular machinery. They offer a more stable and long-term RNAi effect, as they can integrate into the genome and maintain their expression. They are also suitable for creating stable cell lines or transgenic animals with gene silencing. However, they also have some drawbacks, such as potential off-target effects, toxicity, or immune responses. You should carefully select the shRNA sequence, promoter, and vector to avoid these issues.

3 CRISPR interference

CRISPR interference (CRISPRi) is a newer and more precise RNAi technique that uses a deactivated Cas9 protein (dCas9) and a guide RNA (gRNA) to target specific DNA regions and block their transcription. Unlike siRNAs or shRNAs, CRISPRi does not degrade the mRNA, but rather prevents its synthesis. This can reduce the noise and variability of gene expression and allow for reversible and tunable gene silencing. However, CRISPRi also requires efficient delivery and expression of the dCas9 and gRNA components, which can be challenging for some cell types or tissues. You should also verify the specificity and efficiency of your gRNA and dCas9 constructs.

4 RNAi screening

RNAi screening is a high-throughput method to identify genes that are involved in a certain biological process or phenotype. It involves applying a library of siRNAs or shRNAs that target thousands of genes to a cell population and measuring the effects of gene knockdown on a specific readout, such as cell viability, proliferation, or signaling. RNAi screening can help you discover novel gene functions, pathways, or drug targets. However, it also requires careful experimental design, optimization, and validation to ensure the quality and reliability of the results. You should also use appropriate controls, normalization, and analysis methods to reduce false positives and negatives.

5 RNAi tools and resources

To help you find the best RNAi tools for your biotech lab, you can use some of the online databases and platforms that provide information, guidance, and services for RNAi experiments. For example, you can use the RNAi Codex to search for siRNA and shRNA sequences, the CRISPRi Database to find gRNA sequences and data, or the RNAi Consortium to access RNAi libraries and protocols. You can also use the RNAi Central to learn more about the principles and applications of RNAi, or the RNAi Core to outsource your RNAi experiments to experts.

6 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?