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Increasing Throughput in RNA Analysis

Mon, 10/08/2012 - 11:03am
Mike May, PhD, Contributing Editor
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RNA analysis takes an increasingly central role in drug research. Consequently, researchers need more powerful tools to obtain and study these molecules. (Image: Nicolle Rager Fuller, National Science Foundation)  

As healthcare evolves around the world, pharmaceutical companies face new reasons to increase the throughput of RNA analysis. “A lot of the time, our customers in drug research are trying to characterize the response to some type of treatment, like the effects of siRNA or small molecules,” says Richard Fekete, PhD, senior manager in R&D, Life Technologies (Carlsbad, Calif.). “Whether it is cell lines or blood samples, researchers long for cost-effective, higher-throughput methods”.

“Many current technologies for RNA preparation are not well suited for high throughput,” says Fekete. “For instance, to purify RNA from blood you might need to start with 10 milliliters of stabilized blood, and it’s a very manual process for each tube.” With Life Technologies’ Stabilized Blood-to-CT Nucleic Acid Preparation Kit for qPCR, a researcher can start with whole blood stabilized in something like the Tempus Blood RNA tubes and prepare a lysate that can go directly into reverse transcription and real-time PCR. “You can run this in 96-well plates, and in one hour go from stabilized blood to being ready for reverse transcription,” Fekete says.

When needed, a drug researcher can also use the MagMAX for Stabilized Blood Tubes RNA isolation kit to purify the total RNA from blood collected in Tempus tubes. “This magnetic bead-based technology captures microRNA and mRNA at much higher throughput than filter-based approaches,” says Fekete. Like the Blood-to-CT kit, the MagMAX kit also works with 96-well plates. “Once purified, all of the RNA can be analyzed in depth with next generation sequencing or microarray analysis,” Fekete says.

These tools help drug researchers go from screening a huge number of samples down to in-depth analysis of just a few. As Fekete explains: “If you have 100 compounds and you’re testing them in quadruplicate, that’s 400 animals.” Even drawing a daily blood sample over a week creates 2,800 samples. “With our RNA-analysis tools, you can do the initial screen with Blood-to-Ct by qPCR and then go back and purify the RNA of the samples you’re really interested with the MagMAX kit,” says Fekete. “This pure RNA is ready for next generation sequencing to see what is taking place at the transcriptome level.”

Collection to quantification

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This graph shows GoTaq 2-Step RT-qPCR synthesizing cDNA in reactions containing input human reference total RNA that varied over a 9-log range. The insets show the standard curve using the various template amounts in each replicate qPCR (top), and the dissociation curve shows the specificity of GoTaq qPCR reactions (bottom). (Source: Promega Corp.)  

“RNA analysis is used widely across the drug discovery process,” says Amy Hendricksen, global product manager, genomics at Promega Corp. (Madison, Wis.). “We are trying to define diseases at a molecular level, and transcriptomics plays a huge role in that.” Nonetheless, Hendricksen points out the labile quality of RNA. “Obtaining intact RNA can be a challenge for very sensitive downstream applications,” she adds.

Drug researchers can choose from Promega’s manual and semi-automated RNA purification systems: ReliaPrep Systems and the Maxwell 16 Instrument and purification systems, respectively. “The ReliaPrep Systems are a good choice for those looking to purify high-quality RNA from either cultured cells or FFPE [formalin-fixed, paraffin-embedded] tissues,” says Hendricksen. “Our Maxwell simplyRNA purification kits offer rapid RNA purification from blood, cells, or tissue with minimal hands-on time and high reproducibility.”

With purified RNA in hand, drug researchers need to quantify it. “An overestimation of RNA quantity or quality can cause downstream applications to fail,” Hendricksen says. “Promega offers the QuantiFluor RNA system for a rapid, cost-effective method for quantitating RNA samples prior to entering downstream applications.” Plus, Hendricksen points out that Promega’s GoTaq amplification family products—such as the GoTaq 1-Step RT-qPCR, GoTaq 2-Step RT-qPCR, and GoScript reverse transcription systems—“offer higher sensitivity than other systems on the market and are extremely amenable to high-throughput set-up.”

About the author
Mike May is a publishing consultant for science and technology based in Austin, Texas.

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