Mini-RNA Isolation on a Large-Scale MagneSil® Total RNA mini-Isolation System By Terri Grunst, B.S., Promega Corporation The Total RNA mini-Isolation System provides a high-throughput 96- or 384-well format for fast, simple preparation of intact, purified total RNA from small amounts of cultured cells, tissue, or fresh whole blood samples. Total RNA isolation is achieved without the need for vacuum filtration, centrifugation or precipitation. We have developed a new total RNA isolation system that enables high-throughput total RNA isolation without the need for centrifugation, vacuum filtration or precipitation. Procedure The MagneSil® Total RNA mini-Isolation System uses the MagneSil® PMP technology. After sample lysis, nucleic acids are captured by MagneSil® PMPs. The PMPs are then washed and incubated with DNase to remove contaminating genomic DNA. The DNase is inactivated, the degraded genomic DNA is washed away, and the purified total RNA is eluted in Nuclease-Free Water. Sufficient reagents are supplied with the system to isolate total RNA from four 96-well or 384-well plates. All components are guaranteed free of contaminating ribonucleases when used as directed and are thoroughly tested to ensure optimal performance. A summary of the procedure is provided in Figure 1. The automated total RNA isolation procedure takes as little as 30 minutes and includes a DNase step for removal of contaminating genomic DNA. Total RNA can be isolated in 96-well plates from ≤ 1 × 105 cell culture cells, ≤ 2mg of tissue lysate, or ≤ 20µl of whole human blood per well. Total RNA can be isolated in 384-well plates from ≤ 1 × 103 cell culture cells or ≤ 5µl of whole human blood per well. Purified total RNA is suitable for downstream expression profiling amplification reactions such as endpoint and realtime RT-PCR. Average total RNA yield of the MagneSil® Total RNA mini-Isolation procedure is comparable to automated vacuum filtration procedures but provides the advantages of scalability to 384-well format and flexibility of elution volume to maintain purified total RNA sample concentration for downstream applications. Add sample lysate to MagneSil® RNA PMPs. Mix. Capture PMPs. Discard supernatant. Wash PMPs. Incubate PMPs with DNase 10 minutes. Add DNase Stop Solution. Capture PMPs. Discard supernatant. Wash PMPs. We have developed a new total RNA isolation system that enables high-throughput total RNA isolation without the need for centrifugation, vacuum filtration or precipitation. The MagneSil® Total RNA mini-Isolation System(a) chemistry and format is designed for highthroughput total RNA isolation in 96-well or 384-well formats. This system takes advantage of the unique properties of the MagneSil® Paramagnetic Particle (PMP) technology(b) to isolate total RNA from complex biological mixtures. Figure 1. Schematic diagram of the MagneSil® Total RNA mini-Isolation

Table 1. Maximum Amount of Starting Material for the MagneSil® Total RNA mini-Isolation System. Purification of Total RNA From Cell Culture, Tissue Lysate, and Whole Blood Samples The MagneSil® Total RNA mini-Isolation System is designed for the isolation of total RNA from small samples of cultured cells, tissue lysates, or whole blood samples. Isolation of total RNA from tissue lysates and whole blood is demonstrated by endpoint RT-PCR analysis of purified total RNA (Figure 2). Isolation of total RNA from cultured cells is demonstrated by realtime RT-PCR analysis of purified total RNA (Figure...

< Elution Volume (ul) Figure 5. Flexibility in elution volume to control sample concentration. Total RNA isolated from 2 x 104 HeLa cells/well using MagneSil® Total RNA miniIsolation System in a 96-well format. Titration of elution volume shown. Total RNA concentration and yield calculated by measurement of isolated total RNA with Molecular Probes RiboGreen® assay. Sensitivity and Quality of Purified Total RNA Total RNA purified with the MagneSil® Total RNA miniIsolation System was evaluated by realtime RT-PCR analysis. The System is linear over a range of 10 to 105 cells used as starting material...