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Automating RNA Isolation

lity on an Agilent 2100 bioanalyzer. Yields were consistently high (~5 pg per cell for HeLa S3 cells and ~8 pg per cell for K562 cells) and RNA samples were uniformly intact. Furthermore, genomic DNA was effectively removed. An electropherogram demonstrating the high quality of the RNA recovered is shown in Figure 1.

Figure 1.RNA Isolated Using the Automated RNAqueous-96 Protocol. Cultured K562 cells (human leukemia, grown in suspension) were pelleted and resuspended in 1X phosphate buffered saline (PBS), counted and transferred to 48 wells of a deep well 96-well plate at 3.75 x 105 cells/well. Total RNA was isolated using the automated method described at "RNAqueous-96 Automation Protocol", using RNAqueous-96 and a MultiPROBE II HT Liquid Handling System with a Gripper Integration Platform. This RNA sample, which was representative of the 48 samples isolated and analyzed, has a 28S rRNA to 18S rRNA ratio of 1.92, indicating a high level of integrity. This isolation yielded 24 g of RNA (344 ng/l in 70l).

Lack of reproducibility can be an issue with some RNA isolation methods. Real-time RT-PCR was used to assess the reproducibility of the automated RNAqueous-96 procedure. Because real-time RT-PCR is quantitative, reproducible, and can be used to analyze multiple samples simultaneously, it is a good choice for such an analysis. Both an abundant (GAPDH) and a rare target (hTERT: catalytic subunit of human telomerase) were individually analyzed in the previously described HeLa S3 and K562 RNA samples using a TaqMan assay. R


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