We describe automated nucleic acid (NA) isolation from diverse sample types using MagMAX kits (Ambion, Inc.) on KingFisher Magnetic Particle Processors (Thermo Scientific). The MagMAX-96 Blood RNA Isolation Kit is designed for total RNA isolation from whole blood from several species, without white blood cell fractionation, in about 45 min (including genomic DNA removal). The MagMAX-96 Total RNA Isolation Kit is designed for total RNA isolation from up to 2 × 10 6 cultured cells and up to 10-mg tissue. The isolated total RNA is highly intact and pure, ready to use in downstream applications such as microarray analysis or real-time reverse transcription (RT)-PCR for gene expression profiling or pathogen detection. The MagMAX-96 Viral RNA Isolation Kit is designed for viral RNA and DNA isolation from cell free or nearly cell-free samples such as swabs, serum, and plasma; it takes about 15 min. Total NA of high quality and purity is recovered at >75% efficiency, providing high sensitivity for pathogen detection by real-time RT-PCR. Unlike automated liquid handling systems that move reagents into and out of a single well of a multiwell plate to perform the different steps of an RNA isolation procedure, the KingFisher Magnetic Particle Processors use permanent magnetic rods to collect magnetic beads from solution and release them into another well containing reagent for the subsequent step of the procedure. The effectiveness of bead collection and transfer lead to superior washing and elution efficiency, as well as rapid processing. It is a very effective strategy for automation of magnetic-bead-based NA isolation kits. (JALA 2007; 12:195–201)

T he demand for high-throughput RNA isolation has been dramatically increasing with wide applications of RNAi, expression profiling, and molecular diagnosis. A comparison of various RNA isolation methods that have been adapted to high-throughput platforms, focusing on consistently high yield and quality of isolated RNA, reduction of cross-contamination, and simplicity and robustness of the protocol is presented. The streamlining of RNA isolation with RNA quantification by qRT-PCR and amplification for microarray analysis is also discussed. In general, a microspheric bead-based approach results in more consistent RNA recovery than glass fiber filterbased RNA isolation method, and RNA can be eluted in a smaller volume. This is because beads can be fully resuspended in solution to enable more thorough mixing, washing, and elution, whereas the glass fiber matrix is fixed in a filter plate. ( JALA 2004;9:140-5)

W e describe a new technology (patent pending) for high-throughput selection of poly(A) + RNA from total RNA. A novel binding solution is used to ensure the efficient and specific binding of mRNA to oligo(dT) magnetic beads with high stringency, virtually eliminating the non-specific binding of ribosomal RNA (rRNA) either to oligo(dT) beads or to the poly(A) + RNA bound to the beads. As quantified by real-time RT-PCR, more than 99% of the rRNA is removed in a single round selection and mRNAs are fully recovered for both highly-expressed (GAPDH) and poorly-expressed (DDPK) genes from a few µg total RNA. The protocol is adaptable to any generic robotic workstation and takes ~30 minutes to process 96 samples.

W hole blood is an attractive sample source for nucleic acid-based assays because it is readily available, easily accessible, and rich in genetic information. However, globin mRNA accounts for up to 70% of the mRNA (by mass) in whole blood total RNA, resulting in distortion of the RNA amplification and subsequently causing decreased Present calls, decreased call concordance, and increased signal variation in microarray analysis. Therefore, for gene expression analysis, whole blood is typically fractionated before total RNA isolation to reduce globin mRNA content. We have developed a high-throughput sample preparation technology that streamlines workflows for (1) total RNA isolation from whole blood (MagMAX-96 Blood RNA Isolation Kit), (2) globin mRNA removal using a novel, nonenzymatic technology (GLOBINcleardHuman Kit), and (3) mRNA amplification and labeling for expression analysis (MessageAmp II-96 aRNA Amplification Kit). Globin mRNA removal eliminates the need for prefractionation of whole blood, minimizing the potential for expression profile changes during sample handling. Quantitative RT-PCR showed that this method effectively removed up to 95% of the globin mRNA from the isolated RNA while retaining normal levels of other mRNAs. The streamlined sample preparation enables quick and accurate expression analysis of relatively high numbers of blood samples. ( JALA 2006;11:381-6) INTRODUCTIONIn recent years, gene expression profiling, by means of microarrays, has become a powerful, frequently used tool for studying human diseases and their responses to drug therapies. 1 The emergence of pharmacogenomic and personalized medicine in the near future is projected to increase demand for use of this technology. Whole blood is an ideal sample source for gene expression profiling because it is readily available, easily accessible, and rich in genetic information. 2 A robust, automatable process for preparing RNA from whole blood is needed to enable high-volume studies.Most useful genetic information in whole blood resides primarily in the peripheral blood mononuclear cells, which make up approximately 0.1% of the blood cellular fraction. Globin mRNA from reticulocytes accounts for up to 70% (by mass) of the total mRNA in the whole blood. When total RNA from whole blood is amplified using the Eberwine linear amplification procedure, 3 globin mRNA product dominates the amplified RNA (aRNA). This has been shown to decrease Present calls and call concordance, and to increase signal variation. 2,4e6 For this reason, blood is typically fractionated before total RNA isolation. However, fractionation increases sample handling, which can lead to RNA degradation and expression profile changes. Also, blood fractionation is very difficult to automate or to scale up for high-throughput processing because centrifugation is typically used in blood fractionation procedures. For these reasons, an increasing trend is to isolate total RNA from whole blood, followed by globin mRNA reduction before amplification. A typical approac...