Michael Urban, William Strong, and Christina Whitman-Guliaev, Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547 USA

Introduction
The assessment of RNA integrity and purity is an important first step to many gene expression analysis applications. It is preferable to use high-quality, intact RNA as a starting point for applications such as RT-PCR, ribonuclease protection assays,and northern analyses. For some applications, such as cDNA library construction and microarray analyses, which require considerable time and expense, it is essential to qualify the RNA sample before moving forward. Additionally, the stability of RNA transcripts can differ widely throughout the cell, and RNA degradation during extraction and handling can bias the quantitation of transcripts in gene expression studies.
Therefore, a system that provides both sample quality analysis and accurate quantitation adds confidence and value to the results of downstream RNA-based applications.
The Experion automated electrophoresis system uses a combination of Caliper Life Sciences’ innovative LabChip microfluidic separation technology and sensitive fluorescent sample detection to rapidly provide detailed information about RNA sample quality and concentration. Two Experion analysis kits are available for separation and detection of total RNA or mRNA at nanogram and picogram levels. The Experion RNA StdSens (standard-sensitivity) kit is designed for qualitative analysis and quantitation of 5–500 ng/μl total RNA and 25–250 ng/μl mRNA. The Experion RNA HighSens(high-sensitivity) kit is used for qualitative analysis of 100–5,000 pg/μl total RNA and 250–5,000 pg/μl mRNA samples. The Experion system combines the utility and qualitative benefits of gel-based RNA analysis with the quantitative accuracy of spectroscopic analysis.
The Agilent 2100 bioanalyzer is a related microfluidics-based electrophoresis system that uses LabChip technology and
assays that are functionally similar to those used with the Experion system. In this report, we compare the performance of the Experion system and the bioanalyzer in analyzing both total RNA and mRNA samples. Included are a qualitative comparison of sensitivity for both systems and a comparison of the accuracy and reproducibility of RNA quantitation across the dynamic range for each system. The Experion system is demonstrated to provide improved RNA analysis by delivering increased sensitivity for better qualitative assessments and more reliable quantitation of RNA samples.

Methods
Rat brain total RNA and mRNA samples and the 6000 RNA ladder were purchased from Ambion, Inc. The Experion RNA ladder is a component of the Experion RNA StdSens and HighSens analysis kits, which were used for analysis using the Experion system. The RNA 6000 Nano and Pico LabChip kits were used with the Agilent 2100 bioanalyzer and were obtained from Agilent Technologies.RNA samples and ladders were prepared according to the protocols described in the instruction manuals for the Experion RNA analysis kits and the Agilent 6000 LabChip kits. RNA
samples and ladders were first heat-denatured for 2 min at 70°C and then kept on ice until use. The heat-denatured total RNA and mRNA stocks were diluted to the desired final concentrations in either RNase-free TE buffer, pH 7.0 (for use with the Experion RNA StdSens and RNA 6000 Nano LabChip analysis kits), or in DEPC-treated water (for use with the Experion RNA HighSens and RNA 6000 Pico LabChip analysis kits). RNA samples with concentrations ≥5 ng/μl were quantitated spectroscopically by measuring their absorbance at 260 nm, and these concentrations were used to evaluate the quantitation accuracy of both systems. RNA chips were primed, loaded, vortexed, and analyzed
according to the instructions provided with each analysis kit.Each concentration of total RNA or mRNA was analyzed on both systems using a minimum of five chips and three different wells per chip (n = 15 per concentration per RNA type, except for the lowest concentration tested, where n = 10).

Results and Discussion
Qualitative Performance
To minimize any variation in labeling efficiency introduced by differences in sample preparation, both the Experion system and the bioanalyzer employ an RNA stain for detection that binds directly to the RNA moiety. Consequently, the fluorescence intensities of differently sized fragments depend only on the RNA sample concentration and sensitivity of the detection method. Thus, differences in the fluorescence intensity measurements displayed in an electropherogram translate directly into differences in sample concentration. The software in both the Experion system and in the bioanalyzer use the sample fluorescence intensity measurements to calculate quantitative information, such as RNA concentration.
To determine the sensitivity and range of detection of both systems, identical preparations of rat brain total RNA and mRNA at several concentrations were analyzed. The RNA StdSens and RNA 6000 Nano LabChip kits were compared using four concentrations of total RNA (5–500 ng/μl) and mRNA (25–250 ng/μl). The RNA HighSens and RNA 6000 Pico LabChip kits were compared using picogram levels of total RNA (100–5,000 pg/μl) and mRNA (250–5,000 pg/μl). Electropherograms were examined for qualitative differences between the two systems.

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