New Genisphere Assay Amplifies RNA from FFPE up to 1 Million-fold
Genisphere's Bob Getts discusses the company's new target prep kit for expression profiling studies.
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Genisphere has developed a new two-cycle amplification and labeling assay optimized for preparing target RNA isolated from FFPE blocks. The assay amplifies input RNA up to 1 million-fold, enabling whole-genome expression profiling from even a handful of fixed cells. The company’s early collaborations have shown similar expression patterns between fresh frozen and formalin-fixed samples. While some variability exists between the expression data generated from the two sample sets, the underlying biology remains consistent. “In a preliminary experiment we had fresh frozen and then we selected matched FFPE samples for tumor versus normal and we started to do gene expression profiling,” said Bob Getts, Director of R&D, Genisphere. “We validated a series of overlapped signatures in our cancer versus normal study and are moving into a much larger population now.” Many of the company’s early collaborations for RNA profiling from formalin-fixed paraffin-embedded tissue have been with pharmaceutical companies looking to perform gene discovery and validation studies using historical samples with known disease and treatment outcomes. Scientists who want to perform RNA profiling from FFPE samples can use either the SenseAMP or RampUp kits for preparing target. SenseAMP uses a one-cycle amplification, while RampUp uses a two-cycle amplification for lower quantities of input RNA. The target can be used with GeneChip® Human Genome U133 and X3P Arrays; some collaborators have also modified the assay to use with the Affymetrix Human 1.0 ST Exon Array. |
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Getts recently spoke with Affymetrix UserForum editor Tommy Broudy about the company’s new products, performance of expression profiling from FFPE and the types of discoveries being made looking into these samples. TECHNOLOGY AND OPTIMIZATION Getts: The basis of the RampUp and SenseAMP amplification strategies is a random primed reverse transcription process. Round one begins with random and dT primers combined in the reverse transcription. We found very early that the best strategy is to use straight random 9-mers and Oligo dT with nothing really extending on the 5’ end when working with FFPE samples. Anything extra resulted in amplification artifacts later. The RampUp kit uses two rounds of amplification. We’ve created a tandem T7-T3-driven round one, round two amplification, respectively. In round one we use T7, in round two we use T3. During round |
one, the T3 promoter site is automatically integrated into the product. The advantage is that there is no disconnect between rounds that would normally result in lost messages between round one and round two. Further, we found that it’s important to have a greater mass of random primers over the input total RNA sample. If you try to balance or minimize the amount of random primers, you start to have a major impact on amplifying and capturing all messages. The other aspect that we have found is that the ability of reverse transcriptase to reverse transcribe low copy messages is really the weakest point of the overall process. We do add the T4gp32, which has been seen in a variety of different papers to help capture low-level expressers. We see it help for partially degraded samples or more completely degraded samples. The simpler the process—the less manipulations, less transfers, less purification—the more accurate the results. That’s what we found, and we eliminated |
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