These analyses revealed three general trends. First, both the RNA HighSens and RNA 6000 Pico LabChip analyses
correctly identified the ribosomal RNA peaks at all concentrations examined in the picogram concentration series
(data not shown), but only the RNA StdSens analysis kit automatically identified the 18S ribosomal peak at the
5 ng/μl concentration of total RNA (Figure 1A and C) and 25 ng/μl concentration of mRNA (data not shown). Second,
the fluorescence intensities generated by the 18S and 28S peaks in a given sample were, on average, 4–5 times higher
on the Experion system than on the bioanalyzer (Figure 1).
The greater detection sensitivity of the Experion system allows the generation of a higher signal-to-noise ratio; consequently,the consumption of smaller amounts of RNA sample is possible. Third, certain details, such as the presence of small peaks in the 5S region (between the lower marker and 18SRNA) and in the region between the two large ribosomal RNAs, were better resolved above the baseline by the Experion system (Figure 1B and D), potentially leading to better assessment of degraded RNA species or contaminating genomic DNA. By providing additional sample information,
such as the presence or absence of a peak or difference in the intensity of a fluorescent signal over the baseline in identical RNA samples, the Experion system demonstrated greater sensitivity than the bioanalyzer.
Quantitative Performance
In addition to providing a quick visual assessment of RNA quality and integrity, Experion software also performs sample quantitation (calculation of RNA concentration) and provides these results in a Results table. Knowing the sample quality and having accurate and reproducible quantitative data facilitates the successful planning and execution of downstream experiments, such as cDNA construction,microarray analysis, and RT-PCR analysis.
Experion software measures RNA concentration by calculating the area under the electropherogram of an RNA sample and comparing it to that of the RNA ladder, which is provided in the Experion RNA analysis kits at a known concentration. This approach generally yields accurate and reproducible results,though it should be noted that the quality of the RNA ladder preparation and the consistency of chip preparation are also important factors in determining overall quantitation performance. Because the Experion system and the bioanalyzer use similar methods for determining RNA sample concentrations, the accuracy and reproducibility of RNA quantitation with both systems may be directly compared.
Fig. 1. Comparison of sensitivity.Electropherograms were obtained from total RNA separations performed on the Experion system (A and B) and the Agilent 2100 bioanalyzer (C and D) using the nanogram analysis kits. The Experion system’s greater sensitivity is shown by the higher fluorescent signal, which allowed improved sample detection. For 5 ng/μl total RNA, the 18S ribosomal RNA peak was automatically identified by the Experion system software (A),
but not by the bioanalyzer software (C). For 25 ng/μl total RNA, the Experion system also resolved peaks in the 5S region, offering additional RNA sample information (B), while the bioanalyzer did not provide the same level of detail (D).
To determine accuracy, the concentration of each RNA sample was analyzed with both systems and was measured
spectroscopically using UV absorbance at 260 nm.Accuracy, defined as the percent difference between the RNA concentration calculated by the Experion system or the bioanalyzer (chip measurement) and that derived
spectroscopically, was determined using the formula:
[(software concentration – UV concentration)/UV concentration] x 100. Values close to zero indicate parity between the chip and spectroscopic measurements, and a negative or positive value indicates an underestimation or overestimation, respectively, of the chip measurement relative to the spectroscopic measurement.
Reproducibility was evaluated using the coefficient of variation,or CV, as a statistical measure. For each RNA preparation tested, the CV of the concentration reported by the software was determined using the formula: [standard deviation/mean]x 100. CV was expressed as a percentage; small CV values indicate a small degree of variation in replicates and good quantitative reproducibility of the data.
The results of these experiments are summarized in Tables 1 and 2 according to the type of RNA, concentration of RNA,and analysis kit used. The data shown in these tables represent the average measurement of the interchip (across
multiple chips) accuracy and reproducibility for a given concentration and type of RNA.
Overall, the Experion system provided more accurate RNA quantitation than the bioanalyzer in both the nanogram and
picogram range and for both total RNA and mRNA samples.Whereas the RNA StdSens kit displayed a maximum deviation from spectroscopic measurements for total RNA samples of 13.7%, the RNA 6000 Nano LabChip kit produced up to 24.1% deviation for the same set of samples (Table 1).Similarly, the RNA StdSens kit produced measurements within 7.5% of spectroscopic values for mRNA, compared to a maximum 17.0% deviation produced by the RNA 6000 Nano LabChip kit (Table 1). The quantitation accuracy of the RNA HighSens and RNA 6000 Pico LabChip kits was also examined at a single concentration of total RNA and mRNA that could be measured by UV spectroscopy (5,000 pg/μl).
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