Sample preparation is the essential first step to all genomics and transcriptomics, and yet is a primary source of analysis variability and manually intensive. Formalin-fixed, paraffin-embedded (FFPE) samples are routinely collected and hundreds of millions are archived in tissue banks worldwide. These samples are cross-referenced with long-term follow-up patient data crucial to development of therapeutics and biomarker discovery. However, DNA and RNA are currently extracted from FFPE samples using sample preparation techniques which are difficult to automate and accelerate such as wax removal, centrifugation, multiple buffer exchanges, and heated enzyme digestion. Purigen Biosystems proposes a novel system to automate and improve the efficiency of RNA and DNA extraction from FFPE samples. Purigen will develop a protocol and buffer chemistries as well as a novel injection-molded microfluidic chip with eight parallel sample processing channels. Purigen will also modify its existing benchtop system to automate an electric-field-based method for the extraction of DNA and RNA from FFPE tissue. This method is uniquely faster, significantly less manually intensive, and has great potential to achieve higher quality analyses of FFPE tissue. Purigen will use isotachophoresis (ITP) to simultaneously collect, purify, and focus extracted RNA and DNA, quantify total extracted nucleic acid on-chip (via in-line, ITP-aided labeling with an intercalating fluorescent dye), and deliver downstream to eight parallel output reservoirs compatible with robotic pipetting. Purigen will demonstrate the chip and modified system on >80 FFPE samples from colon, breast, liver, and lung tissues, including parallel comparison analyses with traditional methods and quantification of total versus amplifiable yield. Work will include at least 40 runs of fully automated NA extraction for eight samples in parallel in <60 min using Purigen's modified benchtop system, and will demonstrate amplifiable NA yields greater than existing FFPE kits.
For over 100 years, hundreds of millions of tissue samples (many suspected tumors) have been collected and stored in vast, so-called 'biobanks' worldwide. These samples are preserved using a technique known as formalin-fixed, paraffin-embedded (FFPE) tissue preparation. FFPE samples are cross-referenced with unique long-term follow up patient data. The DNA and RNA in these samples therefore hold many possibilities for developing cancer treatments and discovering new disease markers. However, the DNA and RNA are currently extracted from these samples using decades old sample preparation techniques which are difficult to automate and accelerate. Proposed here is a new electric-field-based method of automating the extraction of DNA and RNA from FFPE tissue. This method is faster, significantly less manually intensive, and has great potential to achieve higher quality analyses of all FFPE tissue. The system proposed will leverage low-cost, injection-molded plastic chips and a touch-pad- controlled, automated benchtop device.
