Agilent RNA 6000 Pico Kit Guide

1 Agilent TechnologiesAgilent RNA 6000 PicoKit GuideNotices Agilent Technologies, Inc. 2000-2015, 2016No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or transla-tion into a foreign language) without prior agreement and written consent from Agi-lent Technologies, Inc. as governed by United States and international copyright Part NumberG2938-90046 Rev. CEdition12/2016 Printed in GermanyAgilent TechnologiesHewlett- packard -Strasse 8 76337 WaldbronnWarrantyThe material contained in this docu-ment is provided as is, and is sub-ject to being changed, without notice, in future editions. Further, to the max-imum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a par-ticular purpose.

2 Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or perfor-mance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the sep-arate agreement shall Licenses The hardware and/or software described in this document are furnished under a license and may be used or copied only in accordance with the terms of such Rights LegendIf software is for use in the performance of a Government prime contract or sub-contract, Software is delivered and licensed as Commercial computer software as defined in DFAR (June 1995), or as a commercial item as defined in FAR (a) or as Restricted computer software as defined in FAR (June 1987) or any equivalent agency regu-lation or contract clause.

3 Use, duplication or disclosure of Software is subject to Agi-lent Technologies standard commercial license terms, and non-DOD Departments and Agencies of the Government will receive no greater than Restricted Rights as defined in FAR (c)(1-2) (June 1987). Government users will receive no greater than Limited Rights as defined in FAR (June 1987) or DFAR (b)(2) (November 1995), as applicable in any technical NoticesCAUTIONA CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly per-formed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated condi-tions are fully understood and met. WARNINGA WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly performed or adhered to, could result in personal injury or death.

4 Do not proceed beyond a WARNING notice until the indi-cated conditions are fully understood and Research Use OnlyNot for use in diagnostic procedures3 Contents1 Agilent RNA 6000 Pico Kit42 Required Equipment for RNA 6000 Pico Assay6 Equipment Supplied with the Agilent 2100 Bioanalyzer6 Additional Material Required (Not Supplied)63 Setting up the Assay Equipment and Bioanalyzer7 Setting up the Assay Equipment and Bioanalyzer7 Setting up the Chip Priming Station8 Setting up the Bioanalyzer9 Vortex Mixer9 Starting the 2100 Expert Software104 Essential Measurement Practices115 Preparing the RNA Ladder after Arrival136 Agilent RNA 6000 Pico Assay Protocol14 Agilent RNA 6000 Pico Assay Protocol14 Cleaning the Electrodes before Running Assays14 Preparing the Gel15 Preparing the Gel-Dye Mix16 Loading the Gel-Dye Mix17 Loading the RNA 6000 Pico Conditioning Solution and Marker18 Loading the Diluted Ladder and Samples19 Inserting a Chip in the Agilent 2100 Bioanalyzer20 Starting the Chip Run21 Cleaning Electrodes after a RNA 6000 Pico Chip Run23 Decontaminating the Electrodes247 Checking Your Agilent RNA 6000 Pico Assay Results25 RNA 6000 Pico Ladder Well

5 Results25 RNA 6000 Pico Sample Well Results264 Agilent TechnologiesAgilent RNA 6000 Pico Kit1Ta b l e 1 Agilent RNA 6000 Pico Kit (reorder number 5067-1513) Agilent RNA 6000 Pico Chips25 RNA Pico Chips3 Electrode CleanersAgilent RNA 6000 Pico Reagents (reorder number 5067-1514) (blue) RNA 6000 Pico Dye Concentrate11 This product is provided under a license by Life Technologies Corporation to Agilent Technolo-gies. The purchase of this product conveys to the buyer the non-transferable right to use the pur-chased amount of the product and components of the product only as described in accompanying product literature. The sale of this product is expressly conditioned on the buyer not using the product or its components (1) in manufacturing; (2) to provide a service, informa-tion, or data to an unaffiliated third party for payment; (3) for therapeutic, diagnostic or prophy-lactic purposes; (4) to resell, sell or otherwise transfer this product or its components to any third party, or use for any use other than use in the subfields of research and development, qual-ity control, forensics, environmental analysis, biodefense or food safety testing.

6 For information on purchasing a license to this product for purposes other than described above contact Life Technologies Corporation, Cell Analysis Business Unit, Business Development, 29851 Willow Creek Road, Eugene, OR 97402, Tel: (541) 465-8300. Fax: (541) 335-0354. (green) RNA 6000 Pico Marker (4 vials) (white) RNA 6000 Pico Conditioning Solution (red) RNA 6000 Pico Gel Matrix (2 vials) (yellow) RNA 6000 Pico Ladder (reorder number 5067-1535) (1 vial, 10x concentrate).4 Spin FiltersTu b e s f o r G e l - D y e M i x30 Safe-Lock Eppendorf Tubes PCR clean (DNase/RNase free) for gel-dye mixSyringe Kit1 Syringe51 Agilent RNA 6000 Pico KitTa b l e 2 Physical Specifications TypeSpecificationAnalysis time30 minSamples per chip11 Sample volume1 LKit stability= 4 months at 4 CKit size25 chips11 samples/chip= 275 samples/kitTa b l e 3 Analytical SpecificationsSpecificationTotal RNA AssaymRNA AssayQualitative range50 5000 pg/ L in water250 5000 pg/ L in waterSensitivity (S/N>3)50 pg/ L in water or 200 pg/ L in TE250 pg/ L in water or 500 pg/ L in TEQuantitation reproducibility (within a chip)

7 20 % CV20 % CVQuantitation accuracy30 %-Maximum sample buffer strength50 mM mM EDTAor 50 mM NaCl15 mM MgCl250 mM mM EDTAor 50 mM NaCl15 mM MgCl26 Agilent TechnologiesRequired Equipment for RNA 6000 Pico Assay2 Equipment Supplied with the Agilent 2100 Bioanalyzer Chip priming station (reorder number 5065-4401) IKA vortex mixer Additional Material Required (Not Supplied) RNaseZAP recommended for electrode decontamination (Ambion, Inc. cat. no. 9780) RNase-free water Pipettes (10 L and 1000 L) with compatible tips (RNase-free, no filter tips, no autoclaved tips) mL microcentrifuge tubes (RNase-free).Eppendorf Safe-lock PCR clean or Eppendorf DNA LoBind microcentrifuge tubes are highly recommended. Microcentrifuge ( 13000 g) Heating block or water bath for ladder/sample preparation Mandatory: bayonet electrode cartridge (reorder number 5065-4413)Check the Agilent Lab-on-a-Chip webpage for details on assays: TechnologiesSetting up the Assay Equipment and Bioanalyzer3 Setting up the Assay Equipment and BioanalyzerBefore beginning the chip preparation protocol, ensure that the chip priming station and the bioanalyzer are set up and ready to have t o replace the syringe at the chip priming station with each new kit adjust the base plate of the chip priming station adjust the syringe clip at the chip priming station set up the vortex mixer finally, make sure that you start the software before you load the RNA 6000 Pico assay is a high sensitivity assay.

8 Please read this Guide carefully and follow all instructions to guarantee satisfactory up the Assay Equipment and BioanalyzerSetting up the Chip Priming StationSetting up the Chip Priming Station1 Replace the syringe:aUnscrew the old syringe from the lid of the chip priming the old syringe from the clip. Discard the old the plastic cap of the new syringe and insert it into the it into the hole of the luer lock adapter and screw it tightly to the chip priming the base plate:aOpen the chip priming station by pulling the a screwdriver, open the screw at the underside of the base the base plate and insert it again in position C. Retighten the the syringe clip:aRelease the lever of the clip and slide it up to the top the syringe with each new reagent up the Assay Equipment and Bioanalyzer3 Setting up the BioanalyzerSetting up the Bioanalyzer 1 Open the lid of the bioanalyzer and make sure that the electrode cartridge is inserted in the instrument.

9 If not, open the latch and insert the electrode 1 Electrode cartridge inserted in the instrument (graphic shows an example).2 Remove any remaining Mixer IKA - Model MS31To set up the vortex mixer, adjust the speed knob to 2400 up the Assay Equipment and BioanalyzerStarting the 2100 Expert SoftwareStarting the 2100 Expert SoftwareTo start the software:1Go to your desktop and double-click the following screen of the software appears in the Instrument context. The icon in the upper part of the screen represents the current instrument/PC communication status:2If more than one instrument is connected to your PC, select the instrument you want to use in the tree closed, no chip or chip emptyLid openDimmed icon: no communicationLid closed, chip inserted, RNA or demo assay selected11 Agilent TechnologiesEssential Measurement Practices4 Handle and store all reagents according to the instructions on the label of the individual box.

10 Avoid sources of dust or other contaminants. Foreign matter in reagents and samples or in the wells of the chip will interfere with assay results. Allow all reagents to equilibrate to room temperature for 30 min before use. Thaw samples on ice. Protect dye and dye mixtures from light. Remove light covers only when pipetting. The dye decomposes when exposed to light and this reduces the signal intensity. Always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results. Always wear gloves when handling RNA and use RNase-free tips, microcentrifuge tubes and water. It is recommended to heat denature all RNA samples and RNA ladder before use for 2 min and 70 C (once) and keep them on ice. Do not touch the Agilent 2100 Bioanalyzer instrument during analysis and never place it on vibrating surface.