Conception through build of an automated liquids processing system for compound management in a low-humidity environment.

Boehringer Ingelheim's Automated Liquids Processing System (ALPS) in Ridgefield, Connecticut, was built to accommodate all compound solution-based operations following dissolution in neat DMSO. Process analysis resulted in the design of two nearly identical conveyor-based subsystems, each capable of executing 1400 × 384-well plate or punch tube replicates per batch. Two parallel-positioned subsystems are capable of independent execution or alternatively executed as a unified system for more complex or higher throughput processes. Primary ALPS functions include creation of high-throughput screening plates, concentration-response plates, and reformatted master stock plates (e.g., 384-well plates from 96-well plates). Integrated operations included centrifugation, unsealing/piercing, broadcast diluent addition, barcode print/application, compound transfer/mix via disposable pipette tips, and plate sealing. ALPS key features included instrument pooling for increased capacity or fail-over situations, programming constructs to associate one source plate to an array of replicate plates, and stacked collation of completed plates. Due to the hygroscopic nature of DMSO, ALPS was designed to operate within a 10% relativity humidity environment. The activities described are the collaborative efforts that contributed to the specification, build, delivery, and acceptance testing between Boehringer Ingelheim Pharmaceuticals, Inc. and the automation integration vendor, Thermo Scientific Laboratory Automation (Burlington, ON, Canada).

A highly reproducible, linear, and automated sample preparation method for DNA microarrays.

DNA microarrays are powerful tools to detect changes in transcript abundance in multiple samples in parallel. However, detection of differential transcript levels requires a reproducible sample (target) preparation method in addition to a high-performance microarray. Therefore, we optimized a target-preparation method that converts the poly(A)(+) RNA fraction of total RNA into complementary DNA, then generates biotin-labeled complementary RNA from the cDNA. We measured the efficiency of incorporation of biotin-containing nucleotides by an enzymatic digestion, followed by resolution via analytical high-performance liquid chromatography (HPLC). When the target was hybridized to a sensitive and reproducible microarray platform, low coefficients of variation in both hybridization intensities and differential expression ratios across target preparations were observed. Nearly identical hybridization intensities and expression ratios are observed regardless of whether poly(A)(+)-enriched RNA or total RNA is used as the starting material. We show the ability to discern biological and production variability through the use of different lots of commercial samples as visualized by hierarchical clustering. Automation of the target-preparation procedure shows equivalence to the manual procedure, reproducible yields of target, and low variability as measured by hybridization to microarrays. Most importantly, RNA mixing experiments show a linear and quantitative amplification in probe hybridization signals for >6000 genes across the entire signal range.