RNA interference for gene knockdown and gene silencing
RNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism mediated by non-coding RNA molecules known as microRNAs. microRNAs can target hundreds of genes simultaneous1, inducing subtle but reproducible shifts in gene expression and regulation.
This mechanism can be utilized for targeted gene silencing by introduction of nucleic acid based tools that are specially designed to trigger the RNAi mechanism. These molecules are taken up by the RNA induced silencing complex (RISC) to target specific mRNAs for degradation or to modulate endogenous microRNAs.
Currently, there are three primary categories of RNAi tools to perform these functions:
- small interfering RNA (siRNA)
- short hairpin RNA (shRNA)
- microRNA mimics and inhibitors.
Ectopic gene expression for gene analysis and protein science
Over-expressing genes can offer insight into gene function at multiple levels. Gain-of-function experiments often result in phenotypic changes that provide the counterpart to loss-of-function studies, such as gene knockdown.
Gene expression occurs continuously and simultaneously in cells in order to produce proteins and non-protein coding RNAs, such as rRNA, and tRNA, siRNA. Proteins and RNAs are the functional units of gene expression that catalyze all cellular reactions. Gene expression constructs, such as cDNAs and ORFs, can be used for over-expression studies, enabling a protein of interest to be analyzed for effect on cellular phenotype, intracellular localization, or for the isolation and purification of a protein for further study.
- cDNA is derived from mRNA and contain the ORF as well as all or part of the 5′ and 3′ untranslated regions.
- ORFs are created from cDNA by removing the untranslated regions leaving just the protein coding open reading frame.
CRISPR-Cas9 systems can be used with custom RNA guides for several gene editing applications
Short RNAs can be used to guide protein to targets within complex eukaryotic genomes with high specificity, based upon a naturally occurring system for bacterial and archaeal immunity. Popularly known as CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) technology, these systems rely upon CRISPR-associated (Cas) proteins and have the potential to carry out genome editing functions or alter gene expression.