USER GUIDE. Prelude. Direct Lysis Module PART NOS , 1400-A01

Transcription

1 USER GUIDE Prelude Direct Lysis Module PART NOS , 1400-A01

2 Patents, Licensing and Trademarks NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation and Applause families of products and methods of their use are covered by several issued U.S. and International patents and pending applications ( NuGEN, Ovation, SPIA, Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners. The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right (without the right to modify, reverse engineer, resell, repackage or further sublicense) under these patent applications and any patents issuing from these patent applications to use this product and methods, accompanying this user guide, for research and development purposes solely in accordance with the intended use described and the written instructions provided in this user guide. No license to make or sell products by use of this product is granted to the buyer whether expressly, by implication, by estoppels or otherwise. In particular, the purchase of this product does not include or carry any right or license to use, develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics. For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research, please contact NuGEN Technologies, Inc., 201 Industrial Road, Suite 310, San Carlos, CA Phone or ; FAX or Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase, provided that the product is handled and stored according to published instructions, and that the product is not altered or misused. If the product fails to meet these performance standards, NuGEN will replace the product free of charge or issue a credit for the purchase price. NuGEN s liability under this warranty shall not exceed the purchase price of the product. NuGEN shall assume no liability for direct, indirect, consequential or incidental damages arising from the use, results of use or inability to use its products. NuGEN reserves the right to change, alter or modify any product to enhance its performance and design. NuGEN s products are developed, designed and sold FOR RESEARCH USE ONLY. This product is not to be used for diagnostic or therapeutic purposes, nor is it to be administered to humans or animals. Except as expressly set forth herein, no right to modify, reverse engineer, distribute, offer to sell or sell NuGEN s product is conveyed or implied by buyer s purchase of this NuGEN product. The buyer agrees to use NuGEN products accompanying the product insert in accordance with the intended use and the written instructions provided.

3 Table of Contents Contents I. Introduction... 1 A. Background... 1 B. Lysis Process... 1 C. Performance Specifications... 1 D. Compatibility... 1 E. Quality Control... 1 F. Storage and Stability... 2 G. Material Safety Data Sheet (MSDS)... 2 II. Kit Components... 3 A. Reagents and Supplies Provided... 3 B. Additional Equipment, Reagents and Labware... 3 III. Planning the Experiment... 4 A. Input Sample Requirements... 4 B. Using Nuclease-free Techniques... 4 C. Cell Lysate Storage... 4 IV. Protocol... 5 A. Overview... 5 B. Protocol Notes... 5 C. Cell Lysis Protocol... 5 V. Technical Support... 7 VI. Appendix... 8 A. Use of Lysate in NuGEN RNA Amplification Systems... 8 B. Frequently Asked Questions (FAQs)... 9 C. Update History... 10

4 I. Introduction A. Background The Prelude Direct Lysis Module by NuGEN allows you to prepare cell lysates from isolated cells, cell monolayers or cell culture suspensions for use as input into the following NuGEN RNA amplification kits: Ovation Pico WTA System V2 (Part No. 3302) Ovation PicoSL WTA System V2 (Part No. 3312) Ovation RNA-Seq System V2 (Part No. 7102) B. Lysis Process The Prelude Direct Lysis Module uses a simple cell lysis procedure. The resulting lysate requires no further purification prior to amplification. Cells are collected and pelleted. Then the pellet is washed in the supplied Buffer B and cells are lysed by gentle pipetting in Buffer A, which also inactivates nucleases. The resulting cell lysate is ready for amplification. C. Performance Specifications The cell wash and lysis process is performed in five minutes or fewer and produces cell lysates ready for amplification with a NuGEN RNA Amplification System. D. Compatibility The Prelude Direct Lysis Module is designed for use with the specific NuGEN RNA amplification systems listed above. Please refer to the Appendix for more details or contact a NuGEN Technical Support representative to discuss your assay needs. We do not recommend the use of this protocol on FFPE tissues due to the specific challenges inherent in working with such samples. E. Quality Control Each lot of the Prelude Direct Lysis Module is tested to meet performance specifications. 1 Prelude Direct Lysis Module

5 I. Introduction F. Storage and Stability The Prelude Direct Lysis Module is shipped on dry ice and should be unpacked immediately upon receipt. All components should be stored at 20 C on internal shelves of a freezer without a defrost cycle. After first use, store Buffer A and Buffer B at 4 C. Do not refreeze the buffers. Kits handled and stored according to the above guidelines should perform to specifications for six months after receipt. NuGEN has not yet established long-term storage conditions for the Prelude Direct Lysis Module. G. Material Safety Data Sheet (MSDS) An MSDS for this product is available on the NuGEN website at 2 Prelude Direct Lysis Module

6 II. Kit Components A. Reagents and Supplies Provided Table 1. Prelude Direct Lysis Module Components* COMPONENT PART NUMBER 1400-A01 PART NUMBER VIAL CAP Buffer A S01303 S01305 Clear Buffer B S01304 S01306 Clear *Some components manufactured by Agilent Technologies. B. Additional Equipment, Reagents and Labware Required Materials Equipment Microcentrifuge for individual 1.5 ml and 0.5 ml tubes Microcentrifuge for 0.2 ml individual and 8 X 0.2 ml strip PCR tubes µl pipette, 2 20 µl pipette, µl pipette, µl pipette (Optional) Multichannel pipettes may be useful when working with high numbers of samples in microwell plates. Vortexer Hemocytometer for cell counting Supplies and Labware Nuclease-free pipette tips 1.5 ml and 0.5 ml RNase-free microcentrifuge tubes 0.2 ml individual thin wall PCR tubes or 8 X 0.2 ml strip PCR tubes Disposable gloves Kimwipes Ice bucket 3 Prelude Direct Lysis Module

7 III. Planning the Experiment A. Input Sample Requirements For best results, use cells having a lipid bilayer cell membrane which can be easily pelleted or otherwise removed from culture media (adherent cell cultures, cell suspensions, FACS sorted cells, etc.) As best as possible, estimate the number of cells in your sample. The optimum number of cell equivalents for amplification largely depends upon the total RNA content of the specific cells used, which varies widely depending on cell type and is not always known to any degree of precision. In general, the Prelude Direct Lysis Module can be used with cell inputs in the 50 to 10,000 cell range, again depending upon the RNA content of the specific cell type. Larger numbers of cells can be processed by increasing the volume of Buffer A. Do not exceed 10,000 cells/µl in Buffer A. For more information on this topic please refer to Appendix A of this user guide. Specific RNA input requirements can be found in the user guide associated with the NuGEN RNA Amplification System you intend to use. B. Using Nuclease-free Techniques Nuclease contamination from equipment and work environment can lead to experimental failure. Follow these guidelines to minimize contamination: Wear disposable gloves and change them frequently. Avoid touching surfaces or materials that might introduce nucleases. Use only the reagents provided and recommended. Prior to starting this protocol, clean and decontaminate your work areas and instruments, including pipettes, with commercially available decontamination reagents. Use only new nuclease-free pipette tips and microcentrifuge tubes. C. Cell Lysate Storage Cell lysate produced using the Prelude Direct Lysis Module may be stored at 80 C for up to six months prior to use with minimal freeze/thaw cycles. 4 Prelude Direct Lysis Module

8 IV. Protocol A. Overview Lysate preparation is performed in three quick steps: 1. Count and pellet cells 2. Wash cells 3. Lyse cells B. Protocol Notes Important Note: Due to the high sensitivity of many of NuGEN s RNA Amplification Systems, we recommend carrying out the lysis procedure in a pre-amplification workspace using dedicated pre-amplification consumables and equipment. Wipe all surfaces, equipment and instrumentation with a DNA decontaminant solution such as DNA-OFF (Takara Bio Company Clontech, Cat. # 9036) to avoid the potential introduction of previously amplified cdna into lysates. For more information on our recommendations for workflow compartmentalization and routine lab cleanup please refer to Appendix E of this user guide and to the user guide of the appropriate NuGEN RNA Amplification System. If you have any questions on this important topic, please contact NuGEN Technical Services Always keep thawed reagents and reaction tubes on ice unless otherwise instructed. After thawing and mixing buffers, if any precipitate is observed, re-dissolve it completely prior to use. When instructed to pipet mix, gently aspirate and dispense at least half of total reaction mix volume. Repeat a minimum of 20 times to ensure complete mixing. Components of this NuGEN product should not be used or combined with any other types of Prelude products. C. Cell Lysis Protocol 1. Thaw Buffer A and Buffer B. Vortex to mix and place on ice. Note: Buffer A and Buffer B should be stored at 4 C after first use. 2. Count cells in the sample. 3. Pellet cells by centrifugation at X g for 5 minutes at 4 C. Carefully remove entire volume of supernatant, leaving behind only the cell pellet. 4. Resuspend the cell pellet in 20 µl to 200 µl Buffer B depending on the number of cells in each sample. 5 Prelude Direct Lysis Module

9 IV. Protocol 5. Centrifuge at 200 to 1000 X g for 5 minutes at 4 C. 6. Carefully remove wash solution by aspiration, leaving behind cell pellet. Take care not to disturb the cell pellet. 7. Add a minimum of 1 µl Buffer A per 10,000 cells. When processing large numbers of cells, use greater than 1 µl Buffer A per 10,000 cells. Note: Cell densities higher than 10,000 cells/µl may reduce lysis efficiency and are not recommended. 8. Pipet up and down gently 20 times to mix and lyse cells. Do not vortex the cells in Buffer A. Note: In order to preserve the stability of the RNA, all further dilutions of the lysate prior to amplification should be carried out using Buffer A. 9. Proceed immediately with amplification using a NuGEN RNA Amplification System or freeze the lysate at 80 C for up to six months. Refer to Appendix A for guidance on the use of lysate with NuGEN RNA Amplification Systems. 6 Prelude Direct Lysis Module

10 V. Technical Support For help with any of our products, please contact NuGEN Technical Support at (direct) or , option 2 (toll-free, U.S. only). You may also send faxes to (toll-free) or techserv@nugeninc.com. In Europe contact NuGEN at +31(0) (Phone) or +31(0) (Fax) or europe@nugeninc.com. In all other locations, contact your NuGEN distributor for technical support. 7 Prelude Direct Lysis Module

11 VI. Appendix A. Use of Lysate in NuGEN RNA Amplification Systems Use 1 µl of lysate as input into the NuGEN RNA Amplification System used. Add nuclease-free water as required to make up the remainder of the input volume requirement. For example, if the amplification system protocol calls for 5 µl of sample input, add 1 µl of lysate and 4 µl of nuclease-free water. Desired inputs typically range from 500 pg to 100 ng equivalent total RNA depending on the specific NuGEN RNA amplification system used. Strive to achieve a total RNA input that falls roughly in the range specified for the chosen amplification system. You must estimate this quantity based on the cell count of your sample. The total RNA content per cell of a given type may not be known to any degree of certainty. In these cases, it may be necessary to perform an input titration to test amplification at a number of different inputs. If required, dilute the cell lysate in Buffer A to maintain the RNA stabilizing conditions of the lysate. 8 Prelude Direct Lysis Module

12 VI. Appendix B. Frequently Asked Questions (FAQs) Q1. Which NuGEN amplification systems are recommended for use with the Prelude Direct Lysis Module? The Module has been tested to perform well with the Ovation Pico WTA System V2, Ovation PicoSL WTA System V2 and the Ovation RNA-Seq System V2. Q2. What materials are provided with the Prelude Direct Lysis Module? The Prelude Direct Lysis Module provides a lysis buffer (Buffer A) and wash buffer (Buffer B). Q3. What equipment is required or will be useful? Required equipment includes a microcentrifuge, pipettes, a vortexer and a cell counting device (e.g., hemocytometer). Q4. What additional reagents are required for the Prelude Direct Lysis Module? No additional reagents are required. Q5. What type of cells should I use with the Prelude Direct Lysis Module? Most cell types having a lipid bilayer cell membrane will be compatible with the approach used in this module. Q6. How many cells will I need for amplification? The number of cells required largely depends upon the NuGEN Amplification System you wish to use. Strive to select a cell count that will yield an equivalent mass of total RNA within the recommended input range of the selected amplification system. Q7. Can I vary the number of cell equivalents used for amplification? Yes. As long as the total RNA content of the lysate used is roughly within the range of input recommended by the RNA amplification system chosen, the number of cell equivalents may vary. Q8. Will using lysate impact the expected cdna yield from the amplification reaction? No. In our tests, amplification yields are relatively unchanged when using cell lysate versus an equivalent input of purified total RNA. Q9. Has NuGEN performed reproducibility studies on the Prelude Direct Lysis Module? Yes. Our studies have demonstrated high sample-to-sample, lot-to-lot and operator-to-operator reproducibility. Q10. What are the recommended storage conditions for cell lysates produced using the Prelude Direct Lysis Module? The lysates may be stored at 80 C for up to six months. Ensure the vials are well sealed and minimize freeze/thaw cycles. 9 Prelude Direct Lysis Module

13 VI. Appendix C. Update History This document, the Prelude Direct Lysis Module user guide (M01152 v2) is an update to address the following topics: Description Section Page(s) Updated recommendations for NuGEN amplification kits. Updated information on where to get an MSDS for this product. Removed incorrect information about resuspension of cell pellet in Buffer B in step 4. Updated contact information for NuGEN Technical Support. I.A., VI.B. 1, 9 I.G. 2 IV.C. 6 V. 7 NuGEN Technologies, Inc. Headquarters USA 201 Industrial Road, Suite 310 San Carlos, CA USA Toll Free Tel: Toll Free Fax: custserv@nugeninc.com techserv@nugeninc.com Europe P.O. Box AC Leek The Netherlands Tel: Fax: europe@nugeninc.com For our international distributors contact information, visit our website NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation and Applause families of products and methods of their use are covered by several issued U.S. and International patents and pending applications ( NuGEN, Ovation, SPIA, Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners. For research use only. M01152 v2