USER GUIDE Applause™ WT-Amp Plus ST System PART NO. 5510 Patents, Licensing and Trademarks © 2009–2011 NuGEN Technologies, Inc. The Ovation® and Applause families of products and methods are covered by U.S. Patent Nos. 6,692,918, 6,251,639, 6,946,251 and 7,354,717, and other issued and pending patents in the U.S. and other countries. NuGEN, the NuGEN logo, Ovation, SPIA, Ribo-SPIA, WT-Ovation, Encore, Applause, Prelude 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 prod- uct 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 94070. Phone (888-654-6544 or 650-590-3600); FAX (888-296-6544 or 650-590-3630). 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 drug 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. Table of Contents Contents I. Introduction .........................................................................................................1 A. Background .......................................................................................................1 B. How the Applause WT-Amp Plus ST System Works .........................................1 C. Performance Specifications ...............................................................................3 D. Quality Control .................................................................................................3 E. Storage and Stability .........................................................................................3 F. Material Safety Data Sheet (MSDS) ...................................................................3 II. Kit Components ..................................................................................................4 A. Reagents and Supplies Provided ......................................................................4 B. Additional Reagents, Supplies and Equipment ...............................................5 III. Planning the Experiment .....................................................................................7 A. Input RNA Requirements ..................................................................................7 B. Using RNase-free Techniques ...........................................................................8 C. RNA Storage .....................................................................................................8 D. Amplified cDNA Storage ..................................................................................8 IV. Amplification Protocols .......................................................................................9 A. Overview ...........................................................................................................9 B. Protocol Notes ..................................................................................................9 C. Programming the Thermal Cycler ...................................................................11 D. First Strand cDNA Synthesis ...........................................................................12 E. Second Strand cDNA Synthesis ......................................................................13 F. Post-Second Strand Enhancement..................................................................14 G. SPIA® Amplification ........................................................................................15 H. Post-SPIA Modification I ................................................................................16 I. Post-SPIA Modification II ................................................................................17 J. Purification of ST-cDNA ..................................................................................17 K. Measuring ST-cDNA Yield and Purity ..............................................................19 V. Labeling with the Encore™ Biotin Module .......................................................20 A. Encore™ Biotin Module Overview .................................................................20 B. Protocol Notes ...............................................................................................20 C. Preparing cDNA Samples ..............................................................................20 D. Programming the Thermal Cycler ...................................................................21 E. cDNA Fragmentation .....................................................................................21 F. Biotin Labeling ................................................................................................22 Applause™ WT-Amp Plus ST System Table of Contents VI. Technical Support ...............................................................................................23 VII. Appendix ...........................................................................................................24 A. Target Preparation for Array Hybridization......................................................24 B. Performing Quantitative PCR on Amplified cDNA .........................................25 C. Quality Control of Amplified cDNA Product ...................................................26 D. DNase Treatment of RNA................................................................................27 E. Frequently Asked Questions (FAQs) ...............................................................30 F. Update History ................................................................................................34 Applause™ WT-Amp Plus ST System I. Introduction A. Background The Applause™ WT-Amp Plus ST System provides a fast, simple and cost-effective method for preparing amplified cDNA for global gene expression analysis on Affymetrix® GeneChip® Gene 1.0 ST and Exon 1.0 ST Arrays. Powered by Ribo-SPIA® technology, a rapid, simple and sensitive RNA amplification process developed by NuGEN, the Applause WT-Amp Plus ST System enables the generation of microgram quantities of cDNA in approximately seven hours. Amplification is initiated at the 3´ end as well as randomly throughout the transcriptome, making the Applause WT-Amp Plus ST System an ideal choice for use with whole transcript array designs such as the Gene 1.0 ST and Exon 1.0 ST Array. The Applause WT-Amp Plus ST System (Part No. 5510) provides optimized reagent formulations and a protocol to process total RNA samples. B. How the Applause WT-Amp Plus ST System Works The Applause WT-Amp Plus ST System utilizes Ribo-SPIA technology that produces amplified cDNA from total RNA (see Figure 1). 1. Generation of First Strand cDNA (1 hour) First strand cDNA is prepared from a minimum of 50 ng of high-quality total RNA sample using a unique first strand DNA/RNA chimeric primer mix and reverse transcriptase (RT). The primers have a DNA portion that hybridizes either to the 5´ portion of the poly(A) sequence or randomly across the transcript. RT extends the 3´ DNA end of each primer generating first strand cDNA. The resulting cDNA/ mRNA hybrid molecule contains a unique RNA sequence at the 5´ end of the cDNA strand. 2. Generation of a DNA/RNA Heteroduplex Double-stranded cDNA (1.5 hours) Fragmentation of the mRNA within the cDNA/mRNA complex creates priming sites for DNA polymerase to synthesize a second strand, which includes DNA complementary to the 5´ unique sequence from the first strand chimeric primers. The result is a double-stranded cDNA with a unique DNA/RNA heteroduplex at one end. 3. SPIA® Amplification (1.5 hours) SPIA amplification is a linear isothermal DNA amplification process developed by NuGEN. The process uses a SPIA DNA/RNA chimeric primer, DNA polymerase and RNase H in a homogeneous isothermal assay that provides highly efficient amplifi- cation of DNA sequences. RNase H degrades RNA in the DNA/RNA heteroduplex at the 5´ end of the first cDNA strand. This exposes a DNA sequence that is avail- able for binding to the SPIA DNA/RNA chimeric primer. DNA polymerase initiates replication at the 3´ end of the primer, displacing the existing forward strand. The RNA portion at the 5´ end of the newly synthesized strand is again removed by RNase H, exposing the unique priming site for initiation of the next round of cDNA synthesis. The process of SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated, resulting in rapid accumulation of cDNA with sequence complementary to the original mRNA. 1 Applause™ WT-Amp Plus ST System I. Introduction 4. Post-SPIA Modification, Purification and QC (3 hours) The Post-SPIA Modification process completes the amplification process. The first step allows the random primers to anneal to the single-stranded, antisense cDNA target. The second step utilizes DNA polymerase to extend the annealed primers, producing ST-cDNA targets appropriate for use with GeneChip Gene 1.0 ST and Exon 1.0 ST Arrays. Figure 1. The Ribo-SPIA Whole Transcriptome RNA Amplification Process The Ribo-SPIA® WT-Amp Process 5´ AAAA3´ RNA 3´TTTT 5´ DNA 3´NNNNNN 5´ SPIA Product First Strand cDNA Synthesis RT Reverse Transcriptase NNNNNN WT Primer (DNA/RNA) 5´ AAAA3´ SPIA Primer (DNA/RNA) RT NNNNNN RT TTTT 5´ 5´ TTTT Oligo dT Primer (DNA/RNA) NNNNNNNNN Random 9-mer Second Strand cDNA Synthesis PolDNA Polymerase RT Reverse Transcriptase 35´´ 53´´ Pol DNA Polymerase HRNAseH RNAseH cleavage of RNA Sequence 5´ 3´ 3´ 5´ HRNAseH SPIA Reaction PolDNA Polymerase SPIA Primer Linear Amplification 5´ 3´ Pol Pol Pol Pol 5´ RNAseH Cleavage to Free Primer Hybridization Site 5´ 3´ Pol 5´ HRNAseH Primer Extension by Strand Displacement DNA Synthesis 5´ 3´ Primer Extension by Pol Pol 5´ DNA Polymerase 5´ 3´ Pol 5´ Post-SPIA Modification (ST-cDNA Production) 5´ 3´ SPIA Product NNNNNNNNN Random Primer Pol DNA Polymerase 5´ 3´ Pol NNNNNNNNN Pol NNNNNNNNN 2 Applause™ WT-Amp Plus ST System I. Introduction C. Performance Specifications The Applause WT-Amp Plus ST System synthesizes microgram quantities of ST-cDNA starting with a total RNA input of at least 50 ng. In approximately seven hours, the system produces sufficient cDNA for labeling and subsequent hybridization to GeneChip Gene 1.0 ST and Exon 1.0 ST Arrays. The size of the majority of the cDNA products produced by the amplification process is between 0.1 and 2.0 kilobases. D. Quality Control Each Applause WT-Amp Plus ST System lot is tested to meet specifications of yield and array performance. E. Storage and Stability The Applause WT-Amp Plus ST System is shipped on dry ice and should be unpacked immediately upon receipt. All components should be stored at –20°C in a freezer without a defrost cycle. This product has been tested to perform to specifications after as many as six freeze/ thaw cycles. Kits handled and stored according to the above guidelines will perform to specifications for at least six months. NuGEN has not yet established long-term storage stability for the Applause WT-Amp Plus ST System. F. Material Safety Data Sheet (MSDS) An MSDS for this product is available from NuGEN Technical Service by calling 888-654-6544 or by sending an email to: [email protected]. 3 Applause™ WT-Amp Plus ST System II. Kit Components A. Reagents and Supplies Provided Table 1. First Strand cDNA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER First Strand Primer S01262 Blue A1 ver 4 Mix First Strand Buffer S01256 Blue A2 ver 3 Mix First Strand Enzyme S01040 Blue A3 ver 1 Mix Table 2. Second Strand cDNA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Second Strand S01257 Yellow B1 ver 3 Buffer Mix Second Strand S01126 Yellow B2 ver 2 Enzyme Mix Enhancement S01119 Yellow B3 ver 1 Enzyme Mix Table 3. SPIA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER SPIA Primer Mix S01263 Red C1 ver 8 SPIA Buffer Mix S01258 Red C2 ver 8 SPIA Enzyme Mix S01260 Red C3 ver 5 4 Applause™ WT-Amp Plus ST System II. Kit Components Table 4. Post-SPIA Modification Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Primer Mix S01265 Violet E1 ver 1 Buffer Mix S01266 Violet E2 ver 2 Enzyme Mix S01383 Violet E3 ver 1 Table 5. Additional Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Nuclease-free Water S01001 Green D1 Note: The reagents in the Applause WT-Amp Plus ST System are similar to reagents in NuGEN’s other kits. However, unless the part numbers are identical, these reagents do not have exactly the same composition and, therefore, are not interchangeable. Do not exchange reagents between different kits, as it will adversely affect performance. B. Additional Reagents, Supplies and Equipment Required Materials • Equipment - Microcentrifuge for individual 1.5 mL and 0.5 mL tubes - Microcentrifuge or centrifuge for individual 0.2 mL tubes, strip tubes and PCR plates - 0.5–10 µL pipette, 2–20 µL pipette, 20–200 µL pipette, and 200–1000 µL pipette - Vortexer - Thermal cycler with 0.2 mL tube heat block, heated lid, and 100 µL reaction capacity - Appropriate spectrophotometer and cuvettes, or a Nanodrop® UV-Vis Spectrophotometer • Reagents - Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps 5 Applause™ WT-Amp Plus ST System II. Kit Components • 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, 8 X 0.2 mL strip PCR tubes or 0.2 mL thin wall PCR plates - QIAGEN® MinElute® Reaction Cleanup Kit (Cat. #28204) - Disposable gloves - Kimwipes - Ice bucket Optional Materials • Agilent® 2100 Bioanalyzer or materials and equipment for electrophoretic analysis of RNA • Real Time PCR system • Cleaning solutions such as RNaseZap® (Ambion, Cat.#AM9780) and DNA- OFF™ (MP Biomedicals, Cat.#QD0500) To Order: • Ambion Inc., www.ambion.com • MP Biomedicals, www.mpbio.com • New England BioLabs, www.neb.com • QIAGEN Inc., www.qiagen.com • Sigma-Aldrich, Inc., www.sigmaaldrich.com • USB Corporation, www.usbweb.com 6 Applause™ WT-Amp Plus ST System