Experion RNA StdSens Analysis Kit Instruction Manual

Transcription

1 Experion RNA StdSens Analysis Kit Instruction Manual For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call BIORAD ( )

2 Table of Contents Section 1 Introduction Background Information Product Description Specifications Additional Equipment Requirements Storage Conditions...6 Section 2 Procedure Procedure Overview Essential Practices Cleaning the Electrodes Before a Run Equilibrating Kit Reagents Preparing the Gel Stain Preparing the Samples and RNA Ladder Priming the Chip Loading the Samples and RNA Ladder Into the Chip Running the RNA Analysis Cleaning the Electrodes After a Run...17 Section 3 Data Analysis Example of an RNA Ladder Separation Example of a Total RNA Separation...21

3 3.3 Example of an mrna Separation...23 Section 4 Troubleshooting Electrophoresis and Priming Stations RNA StdSens Analysis...26 Section 5 Ordering Information...28 Section 6 Legal Notices...30 Appendix Quick Guide...31

4 Section 1 Introduction 1.1 Background Information LabChip microfluidic technology provides new opportunities in the development of proteomic and genomic expression analyses. The Experion automated electrophoresis system (Figure 1.1) employs LabChip microfluidic technology to automate electrophoresis for protein and RNA analysis. This microfluidics-based system integrates separation, detection, and data analysis within a single platform. Refer to Appendix D in the Experion automated electrophoresis station instruction manual for additional information on how the Experion system employs this technology. At the center of the Experion system is the microfluidic chip, which contains a series of plastic wells bonded over a small glass plate. The glass plate is etched with an optimized network of microchannels, one of which intersects with each of the plastic wells. Once these channels have been primed with a gel matrix and the samples applied to the appropriate wells, the electrophoresis station directs the samples through these microchannels by controlling the precise voltages and currents that are applied. The microfluidic chip, with the Experion reagents, electrophoresis station, and software, are designed to 1

5 accomplish several sequential procedures, including separation, staining, detection, and basic data analysis without any user intervention a 5b Fig The Experion system includes the following components: (1) Automated electrophoresis station, (2) Priming station, (3) Vortex station for RNA analysis, (4) System operation and data analysis tools, and (5) Analysis kits, which include the (a) chips and (b) reagents for protein (Pro260 kit), standard-sensitivity RNA (StdSens kit), and high-sensitivity RNA analysis (HighSens kit). 2

6 1.2 Product Description Each Experion analysis kit contains the chips and reagents required to perform RNA electrophoresis. Kits are available in 10- and 25-chip configurations. The Experion RNA StdSens analysis kit offers single-step determination of total RNA and mrna purity and concentration at nanogram levels. High-resolution separation and accurate quantitation of the RNA sample is achieved through the combination of chip design innovations and a very accurate RNA ladder, which is included in the kit. The RNA StdSens analysis kit uses a lower internal marker for alignment. The RNA StdSens kit can analyze 1 12 RNA samples in less than 30 minutes. 3

7 RNA StdSens Kit Reagents Item Description Volume/Vial 10-Chip Kit 25-Chip Kit RNA gel Proprietary 1,250 µl 1 vial 2 vials polymer sieving matrix RNA StdSens Proprietary 20 µl 1 vial 2 vials stain fluorescent dye RNA StdSens Contains lower 900 µl 1 vial 2 vials loading buffer alignment marker RNA ladder 20 µl 1 vial 2 vials Spin filters N/A 2 4 RNA StdSens Chips Item 10-Chip Kit 25-Chip Kit RNA StdSens chip 10 chips 25 chips Cleaning chip 2 chips 2 chips 4

8 1.3 Specifications Specification Kit Total RNA mrna Number of wells 12 Sample volume 1 µl Total run time ~30 min Quantitative range ng/µl ng/µl Qualitative range ng/µl ng/µl Maximum sample buffer strength 10 mm Tris, 10 mm Tris, 1 mm EDTA (TE) 1 mm EDTA (TE) 1.4 Additional Equipment Requirements Experion electrophoresis station Experion priming station Experion vortex station Calibrated pipets 0.5 ml microcentrifuge tubes, RNase-free RNase-free aerosol barrier pipet tips Microcentrifuge Heating block or water bath set at 70 C Vortexer RNase-free water (recommend Experion DEPC-treated water, catalog # ) 5

9 1.5 Storage Conditions Item Storage Conditions Shelf Life Experion RNA StdSens reagents 4 C 9 months Experion RNA StdSens chips Ambient 9 months Experion RNA ladder 20 C 9 months Filtered gel (see Section 2.4) 4 C 1 month from filtration Section 2 Procedure 2.1 Procedure Overview 1. Clean electrodes before run. 2. Equilibrate reagents to room temperature. 3. Filter gel. Add stain to filtered gel. 4. Heat RNA ladder and samples. 5. Prime chip with gel-stain solution. 6. Load and vortex chip. 7. Run RNA StdSens analysis protocol on the Experion electrophoresis station. 8. Clean instrument electrodes. 6

10 2.2 Essential Practices Store RNA ladder at 20 C or 70 C. Store all other reagents at 4 C when not in use Before using, allow all kit reagents to equilibrate to room temperature with the exception of the RNA ladder (~15 20 minutes). The RNA stain contains DMSO and should be completely thawed before use Vortex and briefly centrifuge all kit reagents before use Protect the RNA stain and gel-stain solution from light (see Section 2.4). Components in these reagents are light sensitive Always heat-denature the RNA ladder and RNA samples immediately before use Use RNase-free microcentrifuge tubes, pipet tips, and water or TE buffer Avoid sources of dust or other contaminants when preparing samples and loading the chip. Foreign particles in reagents, samples, or in the wells of the chip will interfere with results Always wear gloves when handling the reagents and chips. Handle chips by their edges and do not touch the glass. The chips should only be removed from their packaging immediately prior to use 7

11 Run a loaded chip within 5 minutes of loading. Excessive evaporation may occur, leading to poor results or a chip performance error Do not open the electrophoresis station while running an analysis Never place the electrophoresis station on or near a vibrating surface Dispense reagents into chips slowly. Always insert the pipet tip vertically and to the bottom of the chip well when dispensing liquids. Do not expel air at the end of the pipetting step. This will reduce the possibility of air bubbles becoming trapped between the reagent and the microchannels at the bottom of the chip wells Always follow the recommended electrode cleaning procedure to avoid contamination The use of coated or treated tubes (for example, siliconized polypropylene) should not be used in the preparation of the kit reagents or samples. Use of treated tubes may lead to artifacts during the separation 8

12 2.3 Cleaning the Electrodes Before a Run This procedure should be performed daily. If RNase contamination is suspected, this procedure should be performed between each run. If contamination of the electrodes is still suspected, perform the Electrode Manifold deep Cleaning procedure as described in the protocol provided with the instrument manual. 1. Fill a cleaning chip with 800 µl Experion electrode cleaner. Check to make sure there are no air bubbles trapped in the reservoir. Gently tap the side of the cleaning chip to dispel any bubbles. Label this cleaning chip as the electrode cleaner chip. 2. Open the lid of the electrophoresis station and place the chip on the platform. 3. Close the lid and leave the chip in the instrument for 2 minutes. 4. Fill a separate cleaning chip with 800 µl DEPC-treated water. Label this cleaning chip as the DEPC water chip. 9

13 Note: If this cleaning chip is being used for the first time, treat the chip with the Experion electrode cleaner to remove any RNase contamination prior to use. To do this, completely fill a new cleaning chip with Experion electrode cleaner, let sit for 5 minutes, discard the solution, and then thoroughly rinse (4 5 times) the chip with DEPCtreated water. 5. Open the lid and remove the cleaning chip containing the electrode cleaner; replace it with the chip containing the DEPC-treated water. 6. Close the lid and leave the chip in the instrument for 5 minutes to rinse the electrodes. 7. Replace the DEPC-treated water in the DEPC water chip and repeat the rinse step for 60 seconds. 8. Open the lid and remove the DEPC water chip. 9. Leave the lid open and wait about 60 seconds for any water remaining on the electrodes to evaporate. 10. When finished for the day, remove liquid from the cleaning chips. 10

14 2.4 Equilibrating Kit Reagents 1. Remove the RNA Stain (blue cap), RNA loading buffer (yellow cap), and 1 tube of RNA gel (green cap) from storage and equilibrate to room temperature for ~ minutes. Keep the stain covered at all times to avoid exposure to light. Note: If filtered gel was previously prepared, remove it from storage and equilibrate as detailed above. 2. Vortex the contents of each tube and briefly centrifuge to bring the solutions to the bottom of the tubes. Make sure the DMSO in the stain is completely thawed before proceeding. 2.5 Preparing the Gel Stain Note: Skip steps 1 and 2 if filtered gel is already available. 1. Pipet 600 µl RNA gel (green cap) into a spin filter tube. 2. Centrifuge the gel at 1,500 x g for 10 minutes. Confirm that all of the gel has passed through the filter and then discard the filter. Note: Use the filtered gel within 4 weeks of preparation. After 4 weeks, the filtered gel should be re-filtered and can be reused. 3. Pipet 65 µl filtered gel into an RNase-free microcentrifuge tube. Add 1 µl RNA stain to the tube. Briefly vortex the solution. Keep the gel-stain (GS) solution protected from light. 11

15 This is enough gel-stain solution for 3 chips. Increase the amount of gel stain, if required, by using a 65:1 ratio of gel and stain. Fress gel-stain solution should be prepared daily. 4. Cap the RNA stain tightly, since DMSO is highly hygroscopic, and store it in the dark. 2.6 Preparing the Samples and RNA Ladder 1. Remove the RNA ladder (red cap) from storage and allow it to thaw on ice. 2. Determine the amount of RNA ladder required. A total of 1 µl RNA ladder is required for each chip, plus an extra 1 µl RNA ladder to account for variations in pipeting. As examples, when running 1 chip, 2 µl of RNA ladder will be required; for 3 chips, 4 µl of RNA ladder should be used. 3. Pipet the required amount of RNA ladder into an RNase-free microcentrifuge tube. 4. Prepare all samples by pipetting 2 3 µl sample into RNase-free microcentrifuge tubes. RNA samples should be dissolved in RNase-free water or TE Buffer, as higher ionic strength buffers will effect sensitivity and quantitation accuracy. 5. Denature the ladder and samples for 2 minutes at 70 C. 12

16 6. Cool the denatured ladder and samples by immediately placing the tubes on ice for 5 minutes. 7. Spin down the ladder and samples in a microcentrifuge for 3 5 seconds. Store on ice until used. 2.7 Priming the Chip 1. Open the Experion priming station by pressing down on the front lever. 2. Remove an Experion RNA StdSens chip out of its packaging and place it on the chip platform, matching the arrow on the chip with the alignment arrow on the chip platform. A post on the chip prevents insertion in the wrong position. Do not force the chip into position. 3. Pipet 9 µl filtered gel-stain solution (from Step 2.5) into the well labeled GS (gel priming well). Insert the tip of the pipet vertically and to the bottom of the well when dispensing. Do not expel air at the end of the pipetting step. 13

17 Warning: Placing the pipet tip at the edge of the well or allowing the gel to slide down the wall of the well may lead to bubble formation at the bottom of the well. It is acceptable to allow 1 2 small bubbles at the surface. Dislodge bubbles at the bottom with a clean pipet tip, or remove the gel-stain and refill the well. 4. Carefully close the priming station by gently pressing down on lid. The lid should snap completely closed. 5. Set the pressure setting to B and the time setting to 1, as specified by the alphanumeric code on the chip. 6. Press the Start button. The Priming message will illuminate on the LCD screen, the priming station will pressurize, and the timer will count down. Complete priming requires approximately 30 seconds. Do not open the priming station during the count-down. 7. An audible signal indicates that priming is complete, and a Ready message will be displayed. Open the priming station by pressing down on the release lever. 8. Turn the chip over and inspect the microchannels for bubbles or evidence of incomplete priming. The glass chip will appear opaque and the microchannels will be difficult to see if they are primed properly. If you detect a problem, such as a bubble or incomplete priming, prime a new chip. 9. Place the chip on a clean surface for loading samples. 14

18 2.8 Loading the Samples and RNA Ladder Into the Chip 1. Pipet 9 µl of the gel-stain solution into the other well labeled GS. 2. Pipet 9 µl of filtered gel into the well labeled G. 3. Pipet 5 µl of the loading buffer (yellow cap) in each sample well (1 12) and the ladder well, labeled L. Use a new pipet tip for each delivery to prevent contamination of the loading buffer stock. Alternatively, remove 70 µl of loading buffer into an RNase-free tube and pipet 5 µl to each well from this volume Make sure the pipet tip is centered and positioned vertically all the way to the bottom of the well. Avoid introducing bubbles into the bottom of the wells. It is acceptable to allow 1 2 small bubbles at the surface All wells should be filled with the loading buffer, even when fewer than 12 samples are run. The chip will not run properly unless all wells are filled 15

19 4. Pipet 1 µl denatured RNA ladder into the well labeled L. Every chip must have the RNA ladder loaded into the ladder well for accurate quantitation of samples and for sample alignment 5. Pipet 1 µl sample into each of the 12 sample wells. 6. If running fewer than 12 samples, add 1 µl loading buffer, TE buffer, or DEPC-treated water to the unused sample well(s). 7. Place the chip in the Experion vortex station. 8. Turn on the vortexer, which will operate for 60 seconds and then automatically shut off. Remove the chip when the vortexer stops. 9. Start the run immediately (within 5 minutes) to prevent excessive evaporation and poor results or a chip performance error. 2.9 Running the RNA Analysis 1. Turn on the power to the Experion electrophoresis station by pushing the green button in the center of the front panel. The steady green LED above the button indicates that the unit is on. 2. Launch the Experion software. 3. Open the lid of the electrophoresis station. Place the primed chip, which has been loaded with samples, on the chip platform. Ensure that the chip is seated properly and then carefully close the lid. 4. Select New Run. Select the RNA StdSens protocol (Eukaryotic total RNA, Prokaryotic total RNA, mrna). 16

20 5. Select number of samples to run. Click the Start button to begin the chip run. 6. After a run has started, the green LED in the center of the front panel on the electrophoresis station will begin blinking. 7. When the chip run is complete, a Run complete message will be displayed. Remove the chip from the electrophoresis station and dispose of it properly. To prevent contamination of the electrodes, do not leave the chip in the electrophoresis station for an extended period of time. Also, it is good practice to immediately insert the DEPC water chip (see Step 2.10) as soon as the RNA chip is removed to prevent samples and/or buffers from drying on the electrodes Cleaning the Electrodes After a Run 1. Fill the cleaning chip labeled DEPC water with 800 µl DEPC-treated water. Gently tap the side of the cleaning chip to remove any trapped bubbles from the wells. 2. Open the lid of the electrophoresis station and place the cleaning chip on the chip platform. 3. Close the lid and leave it closed for about 60 seconds. 4. Open the lid and remove the DEPC water chip. 5. Allow the electrodes to dry for seconds. 6. Close the lid. 17

21 7. Replace the water in the cleaning chip after use to avoid contamination. For storage, remove the water from the cleaning chip and store the chip in a clean location. Section 3 Data Analysis Examples of electropherograms generated by the Experion software are provided in this section. For detailed instructions on using the software analysis tools, refer to the software help. 3.1 Example of an RNA Ladder Separation At the end of each run, the Experion software will display an electropherogram of the RNA ladder run in the ladder (L) well of the RNA StdSens chip. Separation of the RNA ladder generates nine peaks in the electropherogram (Figure 3.1a). The software will identify the first peak as the lower alignment marker. If the RNA separation was successful, the other eight RNA peaks in the ladder should be well resolved. 18

22 Fig. 3.1a. Electropherogram of the Experion RNA ladder, illustrating relative positions of the lower marker and RNA peaks. 19

23 Fig. 3.1b. Electropherogram of the Experion RNA Ladder for the mrna assays illustrating the relative positions of the lower marker and RNA ladder peaks. 20

24 3.2 Example of a Total RNA Separation Figure 3.2 shows an electropherogram of a sample of total RNA isolated from rat brain (100 ng/µl) and separated with the Experion RNA StdSens analysis kit. The first peak in the electropherogram corresponds to the lower marker, which is incorporated into the Experion loading buffer and is used for alignment to the RNA ladder. The results shown in Figure 3.2 are typical for separations of total RNA from eukaryotic sources. The two most prominent peaks, aside from the lower alignment marker, represent 18S and 28S ribosomal RNA (rrna). In some cases, depending on the source and method of sample preparation, peaks corresponding to 5S rrna and to trna may also appear (Figure 3.2) Separation of a prokaryotic total RNA sample will appear similar to the electropherogram in Figure 3.2, with the exception that the software will identify the two prominent ribosomal peaks, 16S and 23S. 21

25 Fig Electropherogram of a total RNA sample from rat brain. The relative positions of the lower alignment marker, 18S rrna, and 28S rrna are indicated. 22

26 3.3 Example of an mrna Separation Figure 3.3 shows an electropherogram of a sample of mrna isolated from rat brain (100 ng/µl) and separated using the Experion RNA StdSens analysis kit. Separation of mrna samples will generally generate broad peaks and on occasion, contaminating 18S and 28S rrna peaks will be visible (Figure 3.3). The first peak in the electropherogram corresponds to the lower marker, which is incorporated into the Experion loading buffer and is used for alignment to the RNA ladder. Note that the total analysis time for mrna samples is longer than for total RNA samples (Figure 3.2) in order to include the population of longer mrna transcripts and to achieve a smooth baseline. Also note, the RNA ladder associated with an mrna separation will migrate faster and appear compressed (Figure 3.1b). This is due to higher voltages being used during the analysis as well as the extended run times. 23

27 Fig Electropherogram of mrna sample from rat brain. The relative positions of the lower alignment marker and mrna are indicated. 24

28 Section 4 Troubleshooting For more troubleshooting tips, refer to the Experion electrophoresis station instruction manual. 4.1 Electrophoresis and Priming Stations IV Check Failure message displayed on screen: One or more wells are not filled correctly and the electrode is not completely immersed. All sample wells must be filled, using blanks or replicate samples as necessary Air bubbles are interfering with the electrical contact in one or more of the wells. Tap the chip or use a clean pipet tip to remove or dislodge the bubbles. When pipetting, insert the tip vertically and to the bottom of the well. Dispense the liquid slowly. Do not expel air at the end of the pipetting step Check Seal message lights up: Check or replace the O-ring gasket in the priming station Confirm that there is a chip in the priming station and that the chip is properly positioned on the platform 25

29 4.2 RNA StdSens Analysis No peaks detected in lane on electropherogram: An air bubble was introduced in the sample well or chip by poor priming or sample loading technique. Check the chip for air bubbles after chip priming and sample loading The sample volume in the well is too low. Make sure that 6 µl solution is added to all sample wells. Make sure any unused sample wells contain loading buffer, TE, or DEPC-treated water Particulates are clogging channels. Use high-quality, 0.2 µm filtered water (such as Experion DEPC-treated water) or TE buffer and verify that the gel solution was properly filtered Small peaks in lane on electropherogram: An air bubble is in the sample well. Check the chip for air bubbles after sample loading Ensure that the chip is seated properly in the electrophoresis station chip platform Ensure pipets are calibrated. Use pipets that accurately deliver volumes < 10 µl 26

30 Poor overall chip performance: There is an air bubble in the chip. Check the chip for air bubbles after chip priming Gel was pipetted into the wrong well during priming. Before priming, check to ensure correct loading of gel-stain solution Priming station Replace Seal indicator light is on, but the chip was still used. Check or replace the O-ring Cold reagents were used. Ensure that kit reagents are warmed to room temperature and mixed properly before use Reagents have been degraded. Ensure that the reagents have not expired and light-sensitive reagents have been protected from light Clean the electrode with Experion electrode cleaner to remove any contaminants or RNase Poor ladder and sample separation: The RNA ladder samples, or both were not completely denatured. Make sure the Experion RNA ladder and samples are heated for 2 minutes at 70 C and stored on ice. Do not use RNA ladder that has repeatedly been heated and frozen as this can cause degradation 27

31 If peaks appear degraded, use RNase-free tubes and pipet tips. Do not use autoclaved tubes Section 5 Ordering Information Catalog # Description Experion RNA StdSens Analysis Kit for 10 Chips, includes 10 RNA StdSens chips, 1,250 µl RNA gel, 20 µl RNA StdSens stain, 20 µl RNA ladder, 900 µl RNA StdSens loading buffer, 2 spin filters Experion RNA StdSens Analysis Kit for 25 Chips, includes 25 RNA StdSens chips, 2 x 1,250 µl RNA gel, 2 x 20 µl RNA StdSens stain, 2 x 20 µl RNA ladder, 2 x 900 µl RNA StdSens loading buffer, 4 spin filters Experion RNA StdSens Chips, Experion RNA StdSens Reagents and Supplies for 10 chips, includes 1,250 µl RNA gel, 20 µl RNA StdSens stain, 20 µl RNA ladder, 900 µl RNA StdSens loading buffer, 2 spin filters Experion RNA HighSens Analysis Kit for 10 Chips, includes 10 RNA HighSens chips, 1,250 µl RNA gel, 20 µl RNA HighSens stain, 20 µl RNA ladder, 900 µl RNA HighSens loading buffer, 100 µl RNA sensitivity enhancer, 2 spin filters 28

32 Catalog # Description Experion RNA HighSens Analysis Kit for 25 Chips, includes 25 RNA HighSens chips, 2 x 1,250 µl RNA gel, 2 x 20 µl RNA HighSens stain, 1 x 20 µl RNA ladder, 2 x 900 µl RNA HighSens loading buffer, 2 x 100 µl RNA sensitivity enhancer, 4 spin filters Experion RNA HighSens Chips, Experion RNA HighSens Reagents and Supplies for 10 chips, includes 1,250 µl RNA gel, 20 µl RNA HighSens stain, 20 µl RNA ladder, 900 µl RNA HighSens loading buffer, 100 µl RNA sensitivity enhancer, 2 spin filters Experion Cleaning Chips, Experion Electrode Cleaner, 250 ml Experion DEPC-Treated Water, 100 ml Experion Spin Filters, Experion RNA Ladder, 20 µl 29

33 Section 6 Legal Notices LabChip and the LabChip logo are trademarks of Caliper Life Sciences, Inc. Bio-Rad Laboratories, Inc. is licensed by Caliper Life Sciences, Inc. to sell products using the LabChip technology for research use only. Dye products are licensed under U.S. Patent Nos. US 5,863,753, 5,658,751, 5,436,134 and 5,582,947, and pending patent applications, and related foreign patents, for internal research and development use only in detecting, quantitating and sizing macromolecules, in combination with microfluidics, where internal research and development use expressly excludes the use of this product for providing medical, diagnostic, or any other testing, analysis or screening services, or providing clinical information or clinical analysis, in any event in return for compensation by an unrelated party. 30

34 Appendix Quick Guide Cleaning the Electrodes This procedure should be performed once before the first RNA analysis of the day, or as needed. Place a cleaning chip filled with 800 µl Experion electrode cleaner into the electrophoresis station Close the lid for 2 min. Remove the cleaning chip Place another cleaning chip filled with 900 µl DEPC-treated water into the electrophoresis station Close the lid for 5 min. Replace the water in the cleaning chip and repeat the rinse step. Remove the cleaning chip Equilibrate Kit Reagents Remove RNA StdSens Stain, gel, and loading buffer from storage and equilibrate to room temperature Vortex the contents of each tube and briefly centrifuge Protect stain from light at all times 31

35 Prepare the Gel-Stain Solution Centrifuge 600 µl RNA gel (green cap) in a spin filter at 1,500 x g for 10 min Note: Unused, filtered gel may be stored at 4 C for 1 month Pipet 65 µl filtered gel into an RNase-free microcentrifuge tube. Add 1 µl RNA stain (blue cap) to the filtered gel. Vortex Prepare the RNA Samples and Ladder Pipet at least 2 µl RNA ladder (red cap) and RNA samples into RNasefree microcentrifuge tubes. Denature the ladder and samples for 2 min at 70 C Cool the ladder and samples by placing the tubes immediately on ice for 5 min Briefly spin down the tubes and then mix before use. Store on ice 32

36 Priming the Chip Remove an RNA StdSens chip from its packaging and place it on the chip platform in the Experion priming station Add 9 µl gel-stain solution into the well labeled GS (gel priming well). Close the lid of the priming station, set the pressure to B, and set the time to 1 Press the Start button After priming is finished, remove the primed chip from the priming station and visually inspect the microchannels for trapped air bubbles or incomplete priming Load the Sample and RNA Ladder Into the Chip Pipet 9 µl gel-stain solution into the other well labeled GS Pipet 9 µl filtered gel into the well labeled G Pipet 5 µl loading buffer (yellow cap) into each sample well (1 12) and into the ladder well, labeled L. Do not leave any sample well empty Pipet 1 µl denatured RNA ladder into the well labeled L Pipet 1 µl denatured sample into each of the 12 sample wells. Pipet 1 µl TE buffer or DEPC-treated water in any unused sample wells Place the chip in the Experion vortex station. Vortex for 1 min 33

37 Run the chip in the Experion electrophoresis station within 5 min of loading Running the RNA StdSens Analysis Turn on power to the Experion electrophoresis station and launch the Experion software Place the loaded chip into the electrophoresis station chip platform and close the lid Select New Run and then RNA StdSens. Select the desired RNA assay (Eukaryotic or Prokaryotic Total RNA, or mrna) Click the Start button in the software to begin the run. Select the number of samples to run After the run is complete, remove and discard the used chip Cleaning the Electrodes After a Run Place a cleaning chip filled with 800 µl DEPC-treated water into the electrophoresis station Close the lid for 60 sec to clean the electrodes, and then open it for 60 sec to allow the electrodes to completely dry Remove the cleaning chip 34

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