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