One of the most dynamic areas of research is the study of RNA interference (RNAi). This is a huge field when you consider all the different aspects such as gene silencing and miRNA research. “It’s very complicated, but there is a lot of potential in it,” explains Martin Kreutz, Ph.D., R&D scientist, miRNA research at Qiagen.
“Qiagen has developed a robust and accurate method for transcriptome-wide miRNA quantification using SYBR Green detection-based, real-time PCR,” reports Dr. Kreutz, who will be speaking at Select Biosciences “RNAi Europe” to be held in Stockholm later this month. “The miScript System is highly specific, sensitive, and requires very small amounts of input RNA,” he adds.
The miScript System is a three-component system that comprises the miScript Reverse Transcription Kit, miScript SYBR Green PCR Kit, and miScript Primer Assay. The miScript Reverse Transcription Kit enables reverse transcription of miRNAs, mRNAs, and other noncoding RNAs into cDNA in a single step. The miScript Primer Assay used in combination with the miScript SYBR Green PCR Kit enables real-time PCR of miRNA using SYBR Green detection.
“In contrast to other systems, the cDNA generated using the miScript Reverse Transcription Kit can be used for detection of multiple miRNAs,” says Dr. Kreutz. “A single cDNA preparation can be used with different miRNA primer assays for detection of hundreds of miRNAs, allowing comprehensive expression profiling of known miRNAs. Our system is also well suited to accommodate new developments like 3´ heterogeneity.”
The miScript Reverse Transcription Kit also converts other noncoding RNAs and cellular mRNA into cDNA. This allows the detection of noncoding RNAs and mRNAs by real-time PCR using the appropriate primers (e.g., QuantiTect Primer Assays for mRNA detection).
Dr. Kreutz notes that one of the most interesting aspects of miScript is that it is not only useful for miRNAs but also other noncoding RNAs. “You are not restricted to working with miRNAs—you can also use these samples for multiple targets,” Dr. Kreutz adds. “You can look at a miRNA and its potential target mRNA in one sample. If you store these samples at -20ºC, you can even come back and review the cDNA for a miRNA that was not in your focus of interest when creating that cDNA, or a miRNA that was not even described then.”
Targeted Genome Editing
Rational genome engineering in mammalian cells has great potential across multiple research avenues. To capitalize on these opportunities, Sangamo Biosciences and Sigma-Aldrich recently partnered to commercialize the CompoZr™ technology, which enables high-frequency genome editing via the application of designed zinc finger nucleases (ZFNs).
“Drawing from our work with transformed cell lines, primary human cells, and multipotent stem cells, we will present several examples of single, double, and triple gene knockout, as well as targeted gene insertion into native chromosomal loci,” says Trevor Collingwood, Ph.D., manager of strategic development at Sigma-Aldrich.
Within these ZFNs, the DNA-binding specificity of the zinc finger protein determines the site of nuclease action. Such engineered ZFNs are able to recognize and bind to a specified locus and evoke a double-strand break in the targeted DNA with high efficiency and base-pair precision.
“The cell then employs the natural DNA repair processes of either homology-directed repair or nonhomologous end joining to heal the targeted break,” adds Dr. Collingwood. “These two pathways provide the investigator with the ability to provoke three unique outcomes in genome editing—gene correction, gene deletion, and targeted gene addition.”
The ability to design ZFNs to target specific endogenous loci with high precision enables the researcher to insert transgenes at defined locations, or even to tag endogenous genes with fluorescent markers to follow their expression. The speed and efficiency of this process enables multiple genomic changes in the same cell.
“For example,” Dr. Collingwood explains, “a triple gene knockout in CHO cells is a particularly exciting demonstration of the potential for serious custom engineering of cell lines using CompoZr.”
Dr. Collingwood explains that commercial applications of the ZFN technology initially focused on the optimization of cell lines such as CHO cells for enhanced bioproduction of recombinant therapeutics. Activity is now increasing in the areas of functional genomics and cell-based screening.
An important new application of CompoZr is in the field of animal models such as the rat, where existing methods are lacking. But the technology is not limited to mammalian species; recent publications have demonstrated the use of ZFNs for targeted gene knockout in zebrafish. Together, these applications offer new and enabling approaches to animal and cell-based systems for drug screening and toxicology, as well as recreating models of human disease, Dr. Collingwood says.
“The technology is still in its early days, but this is only the beginning,” notes Dr. Collingwood. “Our goal is to increase ZFN design capacity and throughput to the point where CompoZr technology can be applied by the researcher on a genome-wide scale.”