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Total RNA Purification from Cultured Cells Using the ABI PRISM 6700 Automated Nucleic Acid Workstation and Total RNA Lysis Reagents

Abstract

Applied Biosystems has developed new techniques that produce highpurity total RNA from biological sources such as cultured cells. These techniques eliminate the need for post-isolation DNase treatment or other manual handling, and produce a highly pure product, free of PCR inhibitors, that is less prone to degradation.

These new techniques respond to the demand for high-quality, highthroughput total RNA extraction methods posed by real-time quantitative PCR and other array-based assays.

Introduction

The ABI PRISM 6700 Automated Nucleic Acid Workstation is a flexible, fully automated system, designed to dramatically increase the throughput of nucleic acid preparation and reaction setup. In just over one hour, the 6700 workstation produces 96 purified samples of total RNA.

In less than 2.5 additional hours, the 6700 workstation prepares up to four fully assembled, 96-well reaction plates containing reagents, standards and controls for 5' nuclease assay analysis or other nucleic acid-based assays.

Chemistry

Applied Biosystems lysis chemistry uses a unique formulation to effectively lyse cells or tissues. The lysis solution is supplied as a 2X formulation and should always be used at a final concentration of 1X. Lysis occurs almost immediately after the cells are placed in the lysis buffer or a tissue sample is homogenized in it. The reagent inactivates cellular RNases, preventing degradation of the isolated nucleic acid, and keeps genomic DNA in solution, allowing a clean separation of RNA from all other cellular components.

The cellular lysate or tissue homogenate is transferred to a purification tray and the lysate or homogenate is passed through the tray under precisely controlled vacuum conditions. The 96-well tray (800 μL/well) contains a glass fibre m
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