Feasibility Report Assessment of Total RNA Quality and Concentration with the AdvanCE™ FS96 System

Prepared by:

Advanced Analytical Technologies, Inc. 2711 S. Loop Drive, Suite 4150 Ames, IA 50010 515.296.6600 www.aati-us.com

Feasibility Report Results & Report Generated by: Jeremy Kenseth, Steve Siembieda, Wei Wei Date Report Completed: 07/24/2009

Application: High throughput analysis of total RNA quality and concentration by parallel capillary electrophoresis with fluorescence detection using Advanced Analytical’s AdvanCE™ FS96 System. To demonstrate feasibility a commercially available sample of Rat Total RNA was analyzed. Materials: 1. Commercially available Rat Total RNA sample: FirstChoice® Rat Total Liver RNA (Applied Biosystems/Ambion Part #AM7910), 1 mg/mL solution in 1 mM Sodium Citrate pH 6.4 storage buffer. 2. Materials provided by AATI: dsDNA Gel Wide Range 50-2000bp (AATI #DNF-910-0250) dsDNA Inlet Buffer (AATI #DNF-955-1000) Capillary Conditioning Solution (AATI #DNF-975-1000) DI Water, submicron filtered (Millipore) Urea (Sigma) 19mer ssDNA primer (Lower Marker/Internal Standard) GeneRuler™ 100bp DNA Ladder (Fermentas #SM0241) Sample and buffer 96-wellplates

Sample Preparation Procedures: 1. A urea saturated water solution was initially prepared and filtered. To 3 mL of this solution, 9.9 µL of a 100 µM 19mer primer was added to yield a sample diluent containing approximately 2 ng/µL of 19mer lower marker (LM). 2. The total RNA sample was diluted 25X with sample diluent (4 µL into 96 µL) yielding a stock solution of 40 ng/µL RNA. Successive dilutions were performed on the stock solution with diluent to a final total RNA concentration of 1.25 ng/µL.

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Feasibility Report 3. A separate solution with a concentration of approximately 10 ng/µL RNA was prepared from the total RNA sample (2 µL + 198 µL diluent) and placed in seven additional wells across the sample plate to evaluate the capillary-to-capillary reproducibility for quantification. 4. A commercial 100bp DNA ladder was diluted 100X in sample diluent to a final total concentration of 5 ng/µL and run concurrently with the RNA samples. 5. Samples were placed into a 96-well PCR sample plate (20 µL/well) and directly injected into the instrument.

Instrument Procedures: 1. Samples were analyzed on the AdvanCE™ FS96 System equipped with a 96-capillary array of 50 m i.d. and 55 cm effective/80 cm total length. An LED light source provided an excitation wavelength of 470 nm. 2. The capillary array was initially flushed with Capillary Conditioning Solution and dsDNA Gel using the following sequence: Initial Capillary Conditioning Flush Capillary Conditioning Solution – 300 psi – 7 min dsDNA Gel fill – 300 psi – 10 min Between Run Flush Gel push only – 300 psi – 3 min 3. The separation method consisted of the following: 1. -7.5 kV, 60 sec voltage prerun 2. Water dip 3. 30 sec vacuum sample injection 4. -9 kV, 80 min separation

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Feasibility Report Results: Total RNA Sample Figure 1 shows an overlay of results obtained for a 6-point dilution series of the Rat Total RNA standard prepared in the sample diluent following normalization to the lower marker. From the traces the 18S-rRNA and 28S-rRNA species can be clearly resolved as well as numerous other RNA species. A blank lower marker solution is also shown in the overlay for reference. Some minor impurity peaks are observed around the LM but do not interfere with the RNA related sample peaks.

Figure 1. Overlay of Rat Total RNA dilution series after normalization to LM. A plot of the corrected peak area (CPA) ratio of the total RNA peaks to the lower marker peak versus expected total RNA concentration is shown in Figure 3. A linear response was obtained across the concentration range from 40 - 1.25 ng/µL total RNA.

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Feasibility Report

Figure 2. Plot of expected total RNA concentration vs. CPA ratio to LM. Figure 3 shows the result for the Rat Total RNA sample at a final concentration of 1.25 ng/µL. A signal to noise ratio >40 was obtained for the 28S RNA peak when employing a 30 second vacuum injection, demonstrating the sensitivity of the developed method. The instrument requires a minimum sample volume of 20 L per well. Assuming a 1 µL starting volume of sample with a 20X dilution, the result in Figure 3 translates to a starting sample concentration of 25 ng/µL.

Figure 3. Rat Total RNA in sample diluent at a total concentration of 1.25 ng/µL.

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Feasibility Report Seven replicates of a separately prepared solution of Rat Total RNA at an expected concentration of 10 ng/L were placed in different wells across the sample plate to assess the reproducibility and accuracy of quantification. The total concentration of RNA for each sample was calculated by measuring the corrected peak areas of total RNA and lower marker and using the calibration equation from Figure 2. The results are summarized below in Table 1, showing excellent agreement between the expected and measured total RNA concentration and good reproducibility.

Table 1. Measured total RNA concentration of prepared samples using calibration equation.

Figure 4 shows an overlay of the 10 ng/L total RNA sample from the dilution series and a 100bp DNA ladder run in a separate capillary at the same time. This illustrates that it is possible to perform the RNA purity and concentration assay under the same non-denaturing separation conditions and with the same capillary array as the standard dsDNA fragment sizing application. This results in a greatly simplified workflow as compared to other methods requiring separate gels and capillaries for DNA and RNA analysis.

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Figure 4. Overlay of 10 ng/L Rat Total RNA sample and a 100bp DNA ladder run simultaneously in different capillaries of the array. Summary: Advanced Analytical’s AdvanCE™ FS96 System was demonstrated for analysis of total RNA quality and concentration using the same conditions utilized for standard dsDNA fragment sizing applications. A total of 96 different samples can be analyzed in less than 80 min with good resolution of the 18S/28S species and excellent sensitivity employing vacuum sample injection and internal standardization to a lower marker. A sensitivity of 1.25 ng/L can be obtained with a signal to noise ratio >40, translating to only 1 L of sample at a starting concentration of 25 ng/µL with a 20X dilution. The AdvanCE™ FS96 System offers a flexible platform for performing high resolution, high throughput DNA and RNA analysis. The resolution and run time can be further adjusted via changing the separation gel, capillary length/i.d., and applied voltage depending upon the application.

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