By Ben Butkus
Biotech firm ZyGem and pharmacogenomic assay developer Genomic Health each said this week that they have developed methods for extracting all of the RNA from samples for downstream testing and analysis.
ZyGem's method, which is available as a commercial RNA-extraction kit called RNAGem, involves a single-tube, rapid extraction of RNA from lysed cells. ZyGem said that the method can prepare samples in under 20 minutes, or up to ten times faster than existing RNA extraction products.
Meantime, Genomic Health said that it has developed a method to access the entire transcriptome from formalin-fixed, paraffin-embedded tissues using proprietary RNA preparation chemistry. The company has also demonstrated use of the method to prepare samples for gene expression profiling using Illumina's next-generation sequencing technology.
Many commercial kits for extracting and isolating RNA from tissue samples exist. Indeed, almost all companies offering life science research reagents offer some type of RNA-extraction kit.
However, "most kits tend not to give you the complete, whole RNA sample, because they're not extractions," ZyGem CEO Paul Kinnon told PCR Insider this week. "They include columns, filtrations, and purification steps."
ZyGem's method, on the other hand, uses cell lysis and rapid extraction, "which means that you end up with everything that's in the cell," Kinnon said. "That is fairly unique. We compared this to a lot of kits out there ... and we think we've got the advantage with speed, ease of use, and that we're giving you the whole cell content without having to worry about five different downstream steps."
Overall, Kinnon said, ZyGem's scientists and academic collaborators in New Zealand and the US have been able to do extractions in less than 20 minutes, compared with an hour or more for most commercial examples. "And in the best case, if you don’t need to remove DNA, it's less than 6 or 7 minutes," Kinnon said.
The extraction process also yields smaller RNA molecules such as non-coding RNA and microRNA that are often excluded by other methods, ZyGem said.
ZyGem, based in Solana Beach, Calif., said that it developed the kits in response to a growing number of requests from customers for a high-performance RNA extraction kit that was similar to ZyGem's commercial DNA extraction kit.
Typical customer applications include basic biomedical research at universities, research institutes, and pharma and biotech companies, including single-cell analyses and tissue biopsies; and a growing interest in isolating RNA for diagnostic applications.
For the latter application, many users will require a method to extract total RNA from FFPE tissue samples. ZyGem isn't there yet, but Kinnon said that the company will be releasing kits for DNA extraction from FFPE samples later this month; and for RNA extraction soon thereafter. "That will be much more applicable to the clinical area," he said.
The RNAGem Tissue kits will be distributed exclusively by ZyGem's distribution partner VWR in North America and by VWR International in Europe.
In conjunction with its product launch, ZyGem also said that it has opened a European support office in Bemmel, The Netherlands.
FFPE Profiling
Meantime, Redwood City, Calif.-based Genomic Health said this week that it has developed what it calls a "clinically useful" method for whole-genome expression analysis using FFPE tumor specimens.
Details of Genomic Health's method are a little sketchier, as a company spokesperson told PCR Insider this week that the company could not talk about specific chemistry or methods involved in the technique.
However, at the Advances in Genome Biology and Technology conference held last week in Marco Island, Fla., Genomic Health presented results of a study demonstrating the successful whole-genome expression and mutation analysis from FFPE tumor samples using Illumina next-generation DNA sequencing technology.
As explained in the abstract from that study, RNA sequencing studies using RNA extracted from FFPE specimens "is hampered by the presence of highly abundant ribosomal RNA, which is difficult to remove due to extensive RNA degradation."
Genomic Health's scientists used a method for rRNA removal to selectively degrade 18 S and 28 S rRNA and reduced the representation of those transcripts to less than 5 percent of total mapped reads, according to the abstract.
The study compared expression profiles between two estrogen receptor-positive and -negative breast cancer FFPE specimens that were seven to eight years old. The RNA from these types of samples is highly fragmented and available in minute quantities, which previously made it extremely difficult to conduct expression analysis on a genome-wide scale, Genomic Health said.
Genomic Health's scientists were able to extract, purify, and sequence the complete transcriptome from the FFPE tissue. As many as 18 million RNA transcripts from the individual tumor specimens were sequenced resulting in expression profiles and mutation data on more than 25,000 protein-coding genes, as well as tens of thousands of non-coding transcripts, Genomic Health said.
"These results suggest that it may soon be technically and economically feasible to use whole-genome expression analysis in large clinical trials utilizing routine pathology specimens leading to real commercial applications that may ultimately benefit cancer patients," Joffre Baker, Genomic Health's CSO, said in a statement.
"With that goal in mind, we intend to accelerate our research and development efforts to move this new technology into clinical studies," Baker added.