Performance Comparison of the Experion Automated Electrophoresis System

Table 1. Comparison of accuracy* and reproducibility** of quantitation of nanogram levels of RNA. Total RNA and mRNA samples at the indicated concentrations were analyzed with the Experion RNA StdSens analysis kit and the
RNA 6000 Nano LabChip kit using the Experion system or the Agilent 2100 bioanalyzer, respectively.

* Calculated as % difference relative to values determined by UV.
** Calculated as % CV.
*** The 1,000 ng/μl standard is outside the published concentration range for both the Experion system and the bioanalyzer, so the bioanalyzer data are not included.

The differences between the spectroscopically measured concentrations and those obtained using the RNA HighSens
kit were 19.6% for total RNA and 6.8% for mRNA, compared to 67.9% and 81.7% using the RNA 6000 Pico LabChip kit(Table 2). These data demonstrate that the Experion system generally provides concentration measurements that are most similar to those obtained by spectroscopic methods.The Experion system also displayed greater reproducibility than the bioanalyzer when used for RNA quantitation. With total RNA samples, interchip CVs for the RNA StdSens and HighSens kits ranged between 5.3 and 32.9%, while those for the RNA 6000 Nano and Pico LabChip kits ranged between 9.0 and 48.3%.Similarly, the interchip CVs for mRNA samples ranged from 4.9 to 33.4% and from 19.1 to 33.0% for the Experion system and the bioanalyzer, respectively (Tables 1 and 2). For both systems,reproducibility appeared to be higher for total RNA than for mRNA and tended to be the most reproducible at higher RNA concentrations. The total RNA and mRNA quantitation data generated by the RNA 6000 Pico LabChip kit appear to be similar to those generated by the RNA HighSens kit; however,the bioanalyzer consistently and significantly overestimated the concentration of 5,000 pg/μl RNA preparations, as indicated by the accuracy data for these experiments (Table 2).

Table 2. Comparison of accuracy* and reproducibility** of quantitation of picogram levels of RNA. Total RNA and mRNA samples at the indicated concentrations were analyzed with the Experion RNA HighSens analysis kit and
the RNA 6000 Pico LabChip kit using the Experion system or the Agilent 2100 bioanalyzer, respectively.

* Calculated as % difference relative to values determined by UV.
** Calculated as % CV.
*** The Experion system has a sensitivity range with a lower end as low as 100 pg/μl (total RNA) and 250 pg/μl (mRNA). The bioanalyzer specifications are 200 pg/μl (total RNA) and 500 pg/μl (mRNA).

Figures 2 and 3 provide graphic representations of the performance data in Tables 1 and 2 and both the interchip and
intrachip (multiple wells within one chip) variations in accuracy and reproducibility. The distribution of data in these figures clearly illustrates that the chip-to-chip variations in both accuracy and reproducibility were, in general, smaller for the Experion system, regardless of the type of RNA analyzed.

Conclusions
The Experion system’s greater sensitivity of detection provides the capability to automatically acquire additional sample information, such as the presence or absence of a peak, or peak variations in identical samples, for better qualitative assessment of RNA samples. Overall, the Experion system provides RNA concentration measurements that are more accurate, as they are most similar to those obtained by the traditional spectroscopic methods. The Experion system also displays better reproducibility in RNA sample quantitation, as illustrated by the observed small variations in both interchip and intrachip CV values. The Experion system combines two valuable sample assessments, qualitative and quantitative, into a single analytical process, and provides higher sensitivity,better quantitation accuracy, and greater reproducibility for both total RNA and mRNA samples.For more information on the Experion system, see bulletins 5285 and 3174A.

Fig. 2. Scatter plot comparison of quantitation accuracy (A) and reproducibility (B) data for nanogram levels of RNA. Graphical representation of intrachip data ( ●，mRNA; ●, total RNA; average of 3 wells/chip) and interchip data (, mRNA; , total RNA;average of 5 chips), also presented in Table 1. Left panels, data generated by the Experion RNA StdSens analysis kit; right panels, data generated by the RNA 6000 Nano LabChip kit.

Fig. 3. Scatter plot comparison of quantitation accuracy (A) and reproducibility (B) data for picogram levels of RNA. Graphical representation of intrachip data ( ●, mRNA; ●, total RNA; average of 3 wells/chip) and interchip data (, mRNA; , total RNA;average of 5 chips), also presented in Table 2. Left panels, data generated by the Experion RNA HighSens analysis kit;right panels, data generated by the RNA 6000 Pico LabChip kit.