By Ben Butkus
This article was originally published on April 21.
Scientists from Stanford University, Life Technologies, and other organizations have recently published research demonstrating how long non-coding RNAs can modulate cancer invasiveness and serve as potential targets for diagnosis and therapy.
The study used Life Tech's real-time PCR assays for measuring non-coding RNA expression, representing the first major published validation of the technology since it was launched earlier this year, a Life Tech scientist said.
The paper, published April 15 in Nature, describes work leading to the discovery that increased levels of a specific long intervening non-coding RNA, or lincRNA, called HOTAIR, results in reprogramming of epigenetic components of breast tumors, inducing cancer invasiveness and metastasis. Conversely, the scientists discovered that suppression of HOTAIR inhibits cancer invasiveness.
As part of the work, the researchers used Life Tech's TaqMan non-coding RNA assays, launched in January, to screen breast cancer samples in order to identify HOTAIR overexpression.
Specifically, the researchers developed a panel of 96 TaqMan real-time PCR assays targeting 43 lincRNAs across specific gene loci called HOX, and 39 HOX transcription factors across the loci. They used the assays to profile 88 total RNA samples from five normal breast tissue samples, 78 primary breast tumors, and five metastatic breast tumors.
Yulei Wang, a senior staff scientist with Life Tech and co-author on the study, told PCR Insider last week that the assays showed that several non-coding RNAs were overexpressed in metastatic tumor samples, but particularly HOTAIR, which was overexpressed from several hundred-fold to nearly two-thousand-fold in some samples.
This discovery served as the basis for the entire study, in which the researchers subsequently demonstrated the pathway through which HOTAIR contributes to the epigenetic silencing of genes that suppress metastasis.
In an effort to expand its RNA research portfolio, Life Tech's Applied Biosystems group launched the TaqMan non-coding RNA assays in January, along with Pri-miRNA assays designed to measure the activity of primary microRNAs (PCR Insider, 1/21/10).
In terms of lincRNA, Wang said this week that Life Tech has "been watching this new field very closely and have tried to develop assays to enable the field."
When lincRNAs were discovered a few years ago, Wang said, "we were very excited about their discovery, which we know are very prolifically expressed in the genome, but nobody really knows their function. But we really believe these are a new frontier in life science, and we think we can develop tools to enable discovery in this new field. That's how we initially became involved."
Wang added that the development of the non-coding RNA assays essentially coincided with Life Tech's research collaboration with Stanford and others for the purposes of the Nature study.
"We basically used this panel of HOX non-coding RNAs as our development model, and we received a lot of influence from the researchers at Stanford, in terms of what their requirements and specific needs for non-coding RNA were," Wang said. "We [used] that to optimize our product. Actually, this was the first assay we developed in our line of non-coding RNA assays."
One of the research team's top requirements was a high degree of specificity for non-coding RNA. This was particularly important in the recently published study, because many of the non-coding RNAs within the HOX loci are homologous to one other, Wang said.
"They wanted to make sure that what they looked at was what they actually wanted to look at, and not cross-hybridization," she said. "They wanted to make sure the signal is coming from the specific non-coding RNA and not some artifact. And specificity is one of the strengths of the TaqMan technology."
Wang said that the collaborators on the paper had first attempted to use dynamic tiling arrays to assess overexpression of non-coding RNAs from the HOX region. That technique, however, has a "very limited dynamic range," she said.
"They actually … did tiling array screening first, and the [results were] not that impressive," Wang added. "But when they switched to TaqMan arrays, the change [in overexpression] became more than 100-fold," a result that Wang said is due to the high specificity and sensitivity of TaqMan assays.
The study also represents the first major peer-reviewed publication to vet the non-coding RNA assays, she said.
"We didn't cover this in the study, but we did quite a bit of validation before we went into the clinical samples," Wang said. "So we vigorously tested this assay using normal [tissue] samples, where we knew that these non-coding RNAs … had really distinct programming. We knew which should be turned on and off, and we used that to validate [the assays] so then we could go into clinical samples."