It looks like Ambion’s mirVana line of small RNA purification, detection, and analysis products is about to get bigger. Researchers at the company are currently working on developing several new methods to rapidly and efficiently detect small RNAs — without the need for radioactive materials — and are preparing to introduce a new microRNA microarray, RNAi News has learned.
“The idea is to develop high-throughput detection tools that are specific and sensitive, and do not require radioactivity,” Emmanuel Labourier, senior scientist at Ambion, told RNAi News. “Currently, everybody is using radio-labeled probes to detect small RNAs, and we’d like to go away from radioactivity” because it is both unsafe and inefficient.
One of the approaches Labourier and his colleagues are investigating is the use of a labeled protein to directly detect small RNAs such as siRNAs and microRNAs in microplates. Another is a hybridization protection assay wherein an oligonucleotide probe is internally labeled with a chemiluminescent acridinium ester group.
“What we are working on [are] assays that use, for example, 96-well plates,” Labourier said. “So you can do ... 96 assays in one hour instead of two or three days, sometimes one week, [with] … Northern analysis using radioactivity and polyacrylamide gels.”
Ambion’s scientists are also exploring a novel quantitative RT-PCR-based method for small RNA detection. “You cannot do standard RT-PCR on [small RNAs] because they are shorter than PCR primers,” Labourier said. The technology Ambion is working on is designed to result in PCR products that are significantly larger than the miRNA templates in order to allow quantitation with standard methods like TaqMan.
Existing methods to detect small RNAs with PCR “rely on the ligation of an oligonucleotide to the target RNA you want to detect,” Labourier said. “We see some disadvantages to this kind of approach because ligation can be relatively inefficient and sequence specific sometimes. So we are working on a new, direct PCR detection method that does not incorporate this ligation step.”
Labourier declined to provide a timeline for when these three technologies might be ready for commercialization. He noted that while feasibility work has been completed, “there is still a lot of development ongoing. We just got [a one-year, phase I] grant from the NIH [to develop the methods] … so it’s too early to tell.”
Labourier was, however, able to say that Ambion expects to launch a new microRNA-profiling microarray before the end of the year.
The microarray, which uses a process the company has termed miRNArray analysis, involves four steps: RNA is isolated from a tissue or cell sample using Ambion’s mirVana miRNA isolation kit; RNA species shorter than 30 nucleotides are purified using a rapid column, gel electrophoresis method; miRNAs are labeled with Poly (A) polymerase, amine-modified nucleotides and amine-reactive Cy dyes; then the labeled miRNAs are hybridized on a glass slide where miRNA-specific probes have been arrayed.
The arrays are then processed using standard array scanners.
According to an e-mail from Labourier, the process has been “validated in various mouse and human cell lines or organs, whole blood, white blood cells, as well as tumor and normal adjacent tissues from patients with lung, colon, breast, prostate, bladder, thyroid, or pancreas cancer.
“The new methods we are currently developing in R&D will further facilitate and improve siRNA and miRNA analysis,” he added.