QGE-iPLEX. User s Guide. For Research Use Only. Not For Use in Diagnostics

Transcription

1 QGE-iPLEX User s Guide For Research Use Only. Not For Use in Diagnostics

2 MassARRAY QGE-iPLEX Application Guide SEQUENOM's mission is to be the leading provider of genomic systems and knowledge for personalized medicine and the life science industry. Corporate Headquarters European Office Asia Pacific Office 3595 John Hopkins Court San Diego, CA Phone (858) Fax (858) for orders (858) Mendelssohnstrasse 15D, D Hamburg, Germany Phone (+49) Fax (+49) Herston Road Herston, Qld 4006 Australia Phone (+61) Fax (+61) Beijing Representative Office Technology Building, Suite 702B No. 28, Tian-Zhu Rd, Tian-Zhu Airport Industrial Zone A Shunyi District, Beijing, China Zip: Phone (+86) Fax (+86) sequenom.china@sequenom.com SEQUENOM, K.K Kanda Iwamotocho Plaza Building 9F Iwamotocho, Chiyoda-ku Tokyo Japan Phone: (+81) Fax: (+81) sequenom.japan@sequenom.com Copyright All rights reserved. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, or stored in a database or retrieval system, for any reason other than a licensee's internal use, without the prior written permission of SEQUENOM. TERMS and CONDITIONS for using this product may be found in Appendix C. TRADEMARKS SEQUENOM, MassARRAY, SpectroCHIP, and iplex are registered trademarks of SEQUENOM, Inc. hme genotype reaction is a trademark of SEQUENOM, Inc. All other trademarks are the property of their respective owners. MassARRAY QGE-iPLEX Application Guide Doc R2.0, CO

3 Contents Chapter 1 Introduction to Quantitative Gene Expression on the MassARRAY System Introduction to the MassARRAY System MassARRAY Components Other Essential Components Consumables Overview of the MassARRAY QGE Method Chapter 2 RNA Isolation & Reverse Transcription Introduction Isolating RNA Reverse Transcription To perform reverse transcription using random primers Chapter 3 Processing QGE-iPLEX Reactions Important Procedures and Guidelines Real Competitive PCR for QGE Introduction to Real-Competitive PCR Performing Real Competitive PCR for QGE Neutralizing Unincorporated dntps (SAP Reaction) Creating the QGE-iPLEX Reaction (Extend Reaction) Adjusting the Extension Primers Preparing the QGE-iPLEX Reaction Cocktail Thermocycling the QGE-iPLEX Reaction Cocktail Conditioning the QGE-iPLEX Reaction Products Dispensing onto SpectroCHIP Arrays Defining Assays and Plates Acquiring and Analyzing Spectra Normalizing Data Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

4 Appendix A Extension Primer Adjustment Methods Option A: Two-Tier Method Option B: Three-Tier Method Option C: Four-Tier Method Option D: Regression Method Appendix B Automated Liquid Handling Procedures B.1 Daily and Weekly Maintenance Daily Tasks Weekly Tasks B.2 Adding the SAP Enzyme Solution B.3 Adding the QGE-iPLEX Reaction Cocktail B.4 Conditioning: Adding Water to the Sample Plate Appendix C Terms and Conditions Appendix D References MassARRAY QGE-iPLEX Application Guide Doc R2.0, CO

5 Chapter 1 Introduction to Quantitative Gene Expression on the MassARRAY System 1.1 Introduction to the MassARRAY System This manual provides information on the tasks required for quantitative gene expression (QGE) analysis, using the MassARRAY system. QGE analysis on MassARRAY involves the following tasks (see Figures 1 and 2): RNA isolation, and reverse transcription. Real competitive PCR (amplification reaction). Preparation of the QGE-iPLEX extend reaction products. Transfer of QGE-iPLEX reaction products to SpectroCHIP arrays. Defining the setup of assays and plates in the MassARRAY database using QGE AssayEditor and PlateEditor software. Acquisition of spectra using SEQUENOM s mass spectrometer. Analysis of spectral data using QGE Analyzer software. Normalization of data (optional). NOTE This methodology can also be used to quantify amplifications/deletions in genomic DNA. In this case genomic DNA is substituted for cdna as a template and a single-stranded competitor is used in the real competitive PCR. 1,4 Reactions can be in 96 or 384 formats. You may choose to use either a manual or automated liquid handling process. If you are using an automated process, see Appendix B Automated Liquid Handling Procedures for instructions. Automated liquid handling is for 384 format only. MassARRAY Components The MassARRAY system components are: The MassARRAY Liquid Handler, a 96-channel pipetting robot that provides preprogrammed optimized pipetting schemas for all MassARRAY applications. (Only required for automated liquid handling.) The MassARRAY Nanodispenser, which performs rapid sample transfer onto SpectroCHIP arrays. The MassARRAY Analyzer, an automated benchtop MALDI-TOF mass spectrometer specifically designed for genomic applications. Other Essential Components The following components are not provided with the MassARRAY system, but are required for processing QGE-iPLEX reactions. 384-well thermocycler or 96-well thermocycler Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

6 Chapter 1: Introduction to Quantitative Gene Expression on the MassARRAY System 1.1 Introduction to the MassARRAY System Rotator capable of rotating a microtiter plate a full 360º about its long axis Plate centrifuge Tube centrifuge Twelve-channel pipettor and reservoirs Single-channel pipettors Repeater (multistep pipette) Consumables Table 1: Consumables Needed for Processing QGE-iPLEX Reactions on the MassARRAY System SEQUENOM Consumables iplex Complete Genotyping Reagent Set (#10148 or # ) PCR Accessory and Enzyme Set (#11324) iplex Gold Reagent and Chip Kit (#10134 or # ) iplex Gold Reagent Kit (#10136) SpectroCHIP Arrays and Clean Resin kit (#10117 or # ) General Consumables Disposable plate sealing film (Adhesive PCR Film #SP-0027) Adhesive sealing foil (Marsh Biomedical Products, Inc. #AB-0626) Tubes (1.5 and 5 ml) Water (Deionized: HPLC grade) Complete Genotyping Reagent Set 96 (#10158) PCR Accessory and Enzyme Set 96 Reactions (#10162) NA Matrix Liquid Handler-Specific Consumables µl 96-channel tips for the liquid handler (Matrix #5506) Reagent reservoirs (Matrix # ) 96-well, polystyrene microtiter plates (96- Well Plate Vee Bottom, Sarstedt, Inc. # ) Deionized water Reagent-grade isopropanol 70% Water or mild detergent to wipe off deck and work area iplex Gold Reagent Kit 96 Reactions (#10165) SpectroCHIP Arrays and Clean Resin Kit-96 (#10163) Disposable plate sealing film (Adhesive PCR Film #SP-0027) Adhesive sealing foil (Marsh Biomedical Products, Inc. #AB-0626) Tubes (1.5 and 5 ml) Water (Deionized: HPLC grade) NA NA NA NA NA N/A MassARRAY QGE-iPLEX Application Guide 2 Doc R2.0, CO

7 Chapter 1: Introduction to Quantitative Gene Expression on the MassARRAY System 1.2 Overview of the MassARRAY QGE Method 1.2 Overview of the MassARRAY QGE Method The MassARRAY QGE method combines real-competitive PCR (rcpcr) with iplex procedures and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Following the isolation of RNA and reverse transcription, cdna is spiked with a synthetic DNA molecule (competitor), which matches the sequence of the targeted cdna region in all positions except a single base and serves as an internal standard. The two distinct sequences mimic the situation of two different alleles. As a result, tools for primer extension may be applied that are the same as those used for SNP allele frequency analysis. 1,4,5 The cdna/competitor is PCR-amplified and subjected to a post-pcr shrimp alkaline phosphatase (SAP) enzyme treatment to dephosphorylate remaining nucleotides. After inactivation of the SAP, a primer extension cocktail is added. The PCR products from the competitor and cdna now serve as templates for QGE-iPLEX reactions. The primer extension products are desalted through the addition of clean resin and then dispensed on a SpectroCHIP array. The SpectroCHIP array is then analyzed by the MassARRAY analyzer. During mass spectrometric analysis, the peak areas of the distinct mass signals for the competitor and cdna extension products are calculated. The QGE analyzer software plots cdna frequency versus competitor concentration for each assay and sample. cdna concentrations (expressed as LOGEC50 and EC50) are automatically calculated via nonlinear regression analysis and represent the competitor concentration at which the allele frequencies of cdna and competitor are equal (0.50:0.50). 2 For more information see the MassARRAY QGE Software User's Guide (part # 11498). Figure 1 illustrates rcpcr with the iplex reaction for use in QGE. 3,8 This methodology can also be used to quantify amplifications/deletions in genomic DNA. In such a case genomic DNA is substituted for cdna as a template and a single-stranded competitor is used in the rcpcr. 1,4 A general workflow for conducting MassARRAY QGE experiments is shown in Figure 2. As the diagram shows, screening multiplexed assays with representative cdnas (pooled if conserving them) at various dilutions prior to ordering the competitive templates is suggested, to insure that all of the assays are working together in the multiplex reactions, and to determine a viable cdna dilution for the experiment. MassARRAY QGE is a sensitive procedure since it is PCR cycle independent. 1,4 This allows for the dilution and therefore conservation of valuable cdna samples. Failed assays can be redesigned and run in the context of another multiplex, or can be replexed into the existing multiplex. Once a suitable success rate for the multiplex reactions is established, the competitor oligonucleotides are ordered. Next, the EC50 for each cdna are determined. Since the calculation of the number of cdna molecules present in the starting reaction is the goal of the experiment, running multipoint titration curves is suggested to ensure that the EC50 for all transcripts in the reaction can be determined. The number of titration points and the difference in competitor concentrations between points is up to the user and will differ depending on knowledge of the input cdna. In general, a 12-point titration with 1:7 serial dilutions will cover the complete range of transcript amounts per reaction (1-282,000,000 molecules) while providing an accurate EC50. However, titrations with larger or smaller serial dilutions can be employed Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

8 Chapter 1: Introduction to Quantitative Gene Expression on the MassARRAY System 1.2 Overview of the MassARRAY QGE Method successfully (i.e. 6-point titration with 1:10 serial dilutions). See Figure 3 on page 11 to get an idea of various plate formats. If cdna samples are scarce, an effective initial screen can be conducted to determine the population EC50 by pooling a small aliquot of the cdnas. The EC50 from the cdna pool can then be used as a center point for a reduced point titration for each of the individual cdna samples. Ultimately, it is up to the user to decide the number and dilution constant which best fits the experimental model and is the most cost-effective. MassARRAY QGE-iPLEX Application Guide 4 Doc R2.0, CO

9 Chapter 1: Introduction to Quantitative Gene Expression on the MassARRAY System 1.2 Overview of the MassARRAY QGE Method forward primer cdna Real-Competitive PCR 5 C 5 G 3 C G cdna amplicon reverse primer G C competitor amplicon 3 cdna competitor (60-90 bp) SAP Treatment QGE-iPLEX Reaction extension primer C cdna extension primer G competitor Sample conditioning and nanodispensing Mass Spectrometry of Multiplexed Assays Non-normalized QGE Data Calculation of Most Stable Genes, Geometric Mean, and Normalization Factors using genorm Normalized QGE Data. Figure 1: Overview of Quantitative Gene Expression Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

10 Chapter 1: Introduction to Quantitative Gene Expression on the MassARRAY System 1.2 Overview of the MassARRAY QGE Method Figure 2: MassARRAY QGE Workflow MassARRAY QGE-iPLEX Application Guide 6 Doc R2.0, CO

11 Chapter 2 RNA Isolation & Reverse Transcription 2.1 Introduction The MassARRAY QGE method requires amplification of cdna samples to be used in the MassARRAY system. This chapter describes RNA isolation and reverse transcription, to generate first strand cdna samples. NOTE This methodology can also be used to quantify amplifications/deletions in genomic DNA. In this case genomic DNA is substituted for cdna as a template and a single-stranded competitor is used in the real competitive PCR. 1,4 2.2 Isolating RNA Use a preferred method when isolating either total RNA or mrna. The different products generate a wide range of starting material amounts, so determine the starting material and amounts accordingly. There is no limitation on the source of RNA, just that it is of sufficient quality to be reverse transcribed into cdna for amplification with realcompetitive PCR. NOTE Starting material is an important consideration for QGE. It influences integrity, purity, and amount of RNA subjected to the analysis. Special isolation methods may be needed depending on the starting material. 2.3 Reverse Transcription Any standard reverse transcription kit may be used to facilitate reverse transcription, using one of the following priming methods: Random primers Poly T primer Gene-specific primers NOTE The instructions provided here are a general outline for reverse transcription using random primers. Modify the process as needed according to the requirements of the supplier. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

12 Chapter 2: RNA Isolation & Reverse Transcription 2.3 Reverse Transcription To perform reverse transcription using random primers 1. Prepare the RNA/primer mix as described in Table 2. Table 2: RNA/Primer mix Reagent Volume (1 rxn)* Random primers (50 ng/μl) 1 μl RNA (100 ng/μl) 1 μl DEPC-treated water 8 μl Total Volume: 10 μl *For more than one reaction, an overhang should be added to the volumes. See Section 3.1 on page Denature RNA and primers by incubating at 65º C for 5 minutes, and then place the mixture on ice. 3. Prepare the master reaction mix as described in Table 3. Table 3: Master Reaction Mix Reagent Volume (1 rxn)* 5x cdna synthesis buffer 4 μl 0.1 M DTT 1 μl RNaseOUT (40 U/μL) 1 μl DEPC-treated water 1 μl 10 mm dntp mix 2 μl ThermoScript RT (15 U/μL) 1 μl Total Volume: 10 μl *For more than one reaction, an overhang should be added to the volumes. See Section 3.1 on page Add 10 µl of master reaction mix to the RNA/primer mix on ice. 5. Incubate in a thermocycler as follows: 25º C for 10 minutes 50º C for 50 minutes NOTE Store first-strand cdna products at -20º C if not used immediately. MassARRAY QGE-iPLEX Application Guide 8 Doc R2.0, CO

13 Chapter 3 Processing QGE-iPLEX Reactions 3.1 Important Procedures and Guidelines It is important that the following procedures are adhered to throughout the processing of QGE-iPLEX reactions: Gloves and safety glasses must be worn when handling all equipment, components, and reagents. When preparing a cocktail or solution, add reagents in the order in which they are listed in the table. When running more than one reaction, apply an appropriate overhang to reagent volumes. We recommend 20% for manual liquid handling, and 38% (the maximum allowed) for automated liquid handling. After adding reagents to prepare a solution or cocktail, vortex and centrifuge the solution or cocktail before proceeding. Seal and lightly vortex and centrifuge all sample plates before cycling. Due to the high likelihood of evaporation of the small reaction volume in the 96- well plates, we recommend the following thermocyclers: Perkin Elmer: PE7500, PE9700 ABI: GeneAmp PCR System well ( ab?cmd=catnavigate2&catid=600968) Note: We highly discourage the use of MJ Research Tetrad cyclers. When programming the thermocyler for the first time in each step, save and name the programs (for example, QGE rcpcr, SAP, iplex Extend). Then for future reactions, just run the appropriate program. To minimize evaporation use plate seals: Adhesive PCR Film Cs/100 ( shopdisplayproducts.asp?search=yes&sppp=10) Bio-Rad Pressure Pads (#ADR-5001); $15ea ( US/adirect/biorad?cmd=catProductDetail&vertical=LSR&productID=ADR- 5001) Sample plates may be sealed and stored at -20º C for up to 2 weeks after the unincorporated dntps are neutralized, after the QGE-iPLEX reaction is processed, and/or after the samples are conditioned, if not going on immediately to the next step. Be sure to thaw sample plates to room temperature before proceeding. Liquid handling procedures are not described in this chapter, but instructions are given in Appendix B for processing 384-well plates on the MassARRAY Liquid Handler. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

14 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE 3.2 Real Competitive PCR for QGE Introduction to Real- Competitive PCR This section covers real-competitive PCR (rcpcr) which entails the co-amplification of cdna and competitive templates (competitors) to be used for MassARRAY QGE analysis. Each competitor molecule serves as an internal control and matches its target cdna sequence at all nucleotide positions except a single base so the two can be resolved using QGE-iPLEX extension assays. The PCR primers bind and co-amplify the cdna and competitor templates with equal stoichiometry and kinetics. This process preserves the initial cdna: competitor ratio. 1,4,5 Since the cdna concentration of any one transcript is unknown, a titration of competitor concentrations is used to determine the competitor concentration at which amplification between cdna and competitor are equal. 4,6,7,8 This point, termed the EC50, is determined by plotting cdna allele frequency vs. competitor concentration (see QGE Software Guide, part # 11498). Non-linear regression is used to calculate the point at which the cdna and competitor are at a 1:1 ratio (0.5:0.5 allele frequencies). 2 Since the calculation of the number of cdna molecules present in the starting reaction is the goal in QGE, we suggest running multipoint titration curves to ensure that the EC50 for all transcripts in the reaction can be determined. The number of titration points and the difference in competitor concentrations between points is up to the user and will differ depending on the knowledge they have of the input cdna. In general, a 12-point titration with 1:7 serial dilutions will cover the complete range of transcript amounts per reaction (1-282,000,000 molecules) while providing an accurate EC50. However, titrations with larger or smaller serial dilutions can be employed successfully (i.e. 6-point titration with 1:10 serial dilutions) see Figure 3 for examples of different plate layouts. NOTE The procedure for rcpcr can also be used for allele specific expression with, for example, addition of a bi-allelic cdna sample and a competitor template that mimics a third allele in the PCR. See the Application Note Measuring Allele-Specific Expression Using MassARRAY (SEQUENOM, Inc). 6 MassARRAY QGE-iPLEX Application Guide 10 Doc R2.0, CO

15 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE A: 8 samples per plate, 1x10-18 M to M competitor concentration range, plus positive control, 1:7 dilution steps. B: 8 samples per plate, 1x10-18 M to 1x10-8 M competitor concentration range, plus positive control, 1:10 dilution steps. C: 16 samples per plate, 1x10-18 M to 1x10-13 M competitor concentration range, 1:10 dilution steps. D: 24 samples per plate, 1x10-18 M to 1x10-12 M competitor concentration range, 1:100 dilution steps. Figure 3: Plate Layout Examples for QGE Experiments Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

16 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE Performing Real Competitive PCR for QGE The steps in this section cover performing real competitive PCR, using either a 96-well or a 384-well sample microtiter plate format, with 5 µl reactions in each well. The PCR primers and competitive templates for all assays included in the multiplex are added to the same reaction well for each individual cdna and each specific concentration of competitors. Use of at least four replicates for each reaction is recommended in order to obtain the most statistically accurate results, but the actual number of replicates is up to the user. Positive controls with only cdna can also be performed when desired. These serve as the starting point in plotting cdna frequency vs. competitor concentration, yielding a cdna allele frequency of ,5,6,7 Three steps are involved in setting up the PCR sample plate: 1) Preparing the competitor titration dilution series of concentrations, and dispensing them to a 96-well microtiter plate (competitor plate). 2) Preparing the PCR premixes (including cdna), and dispensing them to the final sample plate (96- or 384-well). 3) Adding the competitor concentrations to the PCR premixes in the final 96- or 384-well sample plate. The more detailed instructions that follow use as an example a plate of reactions in which the same assays in a multiplex are applied to sixteen individual cdna samples, using a six-log titration of competitors, either with one replicate per reaction (for the 96-well final sample plate Figure 4A) or four replicates per reaction (for the 384-well final sample plate Figure 4B). Two possible protocols for completing these steps are described below: Protocol A (25 µl reaction dispensed to 5 µl reactions; for a 384-well final sample plate) involves adding 15 µl of PCR premix (including cdna) and 10 µl of competitor mix to each well of a 96-well plate, resulting in 25 µl reactions. Then, 5 µl from each of these wells is transferred to 4 replicate wells in the final 384-well sample plate. This protocol is highly recommended, as it can help reduce errors resulting from pipetting inaccuracies. Protocol B (5 µl reaction; for either a 96- or 384-well final sample plate) involves adding 3 µl PCR premix (including cdna) and 2 µl competitor mix to each sample well in the final sample plate, resulting in a 5 µl reaction. MassARRAY QGE-iPLEX Application Guide 12 Doc R2.0, CO

17 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE A 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 B 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 C 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 D 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 E 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 F 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 G 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 H 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 Figure 4A. 96-well final sample plate Figure 4B. 384-well final sample plate Figure 4: Final Sample Plates Examples of plate layouts for QGE analysis. In these examples, 16 unique cdnas are assayed using a 6-log titration of a competitor template, with one replicate per reaction in the 96-well plate, and 4 replicates per reaction in the 384-well plate. NOTE Preparing the PCR cocktails (including cdna samples) and competitor dilutions in separate hoods is recommended in order to eliminate the risk of contamination of competitor templates into PCR reagents and wild type cdna samples. Also, perform pre- PCR and post-pcr preparations in separate work areas. NOTE MassARRAY QGE is a highly sensitive technology. Dilution of first strand cdna reactions is often required. Prior to running individual samples, we recommend pooling a small aliquot of each sample and testing dilution ranges from 1:10 to 1:1000 to determine the optimal dilution for your cdnas. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

18 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE I) Prepare the competitor titration mixes: 2 µl of competitor mix will be required for each sample well in the final 96-or 384-well sample plate. 1. For the example used here, prepare a multi-step titration of competitor mixes in a 96-well microtiter plate, as shown in Table 4. For Protocol A (25 µl reaction dispensed to 5 µl reactions; for a 384-well final sample plate), 160 µl of each of the six one-log titrations will be needed, plus appropriate overhang. For Protocol B (5 µl reaction; for either a 96- or 384-well final sample plate), 128 µl of each of the six one-log titrations will be needed, plus appropriate overhang. Table 4. Example of 96-Well Microtiter Plate Containing 6-Log Ttitration of Competitor Templates (2.5 x to 2.5 x M) A 2.5x x x x x x10-13 NOTE Competitor concentration varies depending upon experiment design (in this example 2.5x10-18 M to 2.5x10-13 M). The addition of competitor mix gives a 2:5 dilution in the final reaction. The final concentrations in the reaction titration represent the multi-log range. In this example, the final range of competitor concentrations is 1x10-18 M to 1x10-13 M. A competitor mix represents all competitor templates in the multiplex. 2. Seal the 96-well competitor mix plate and store in refrigerator until use (or store below -20 C for use another day; thaw before proceeding). Mix and centrifuge the plate before use in Step 5A or 5B below. II) Prepare the PCR premixes: 3 µl of PCR premix will be required for each sample well of the final 96- or 384-well sample plate. 3. For the example used here, prepare the PCR premix as shown in Table 5. For Protocol A (25 µl reaction dispensed to 5 µl reactions; for a 384-well final sample plate) prepare 90 µl of PCR premix for each of the 16 cdna samples, plus appropriate overhang. For Protocol B (5 µl reaction; 384-well final sample plate) prepare 72 µl of PCR premix for each of the 16 cdna samples, plus appropriate overhang. For Protocol B (5 µl reaction; 96-well final sample plate) prepare 18 µl of PCR premix for each of the 16 cdna samples, plus appropriate overhang. MassARRAY QGE-iPLEX Application Guide 14 Doc R2.0, CO

19 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE Table 5. PCR Cocktail PCR premix Reagent Final Concentration Protocol A Volume of reagent for one 25 μl rxn* (to be dispensed to 4 wells) HPLC grade water N/A 1.50 µl 0.30 µl 10x PCR buffer (with 20 mm MgCl 2 ) 1x (2 mm MgCl 2 **) 2.50 µl 0.50 µl MgCl 2 (25 mm) 2 mm** 2.00 µl 0.40 µl dntp mix (25 mm) 500 µm 0.50 µl 0.10 µl 1 µm PCR primer mix 0.1 µm 2.50 µl 0.50 µl cdna from reverse TBD 5.00 µl 1.00 µl transcription reaction PCR enzyme (5 1.0 Unit/rxn 1.00 µl 0.20 µl Units/ul) Total: µl 3.00 µl Competitor mix or water (from 96-well plate) TBD µl 2.00 µl Protocol B Volume of reagent for one 5 μl rxn* Total: µl 5.00 µl *For more than one reaction, an overhang should be added to the volumes. See Section 3.1 on page 9. **Final concentration of MgCl 2 equals 4 mm in 5.00 µl (2 mm from the PCR buffer and 2 mm from the MgCl 2 NOTE A PCR primer mix represents all primers (forward and reverse) for all templates in the unior multiplex. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

20 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE III) Add the competitor mixes to the PCR premixes (2 µl of competitor mix and 3 µl of PCR premix will be in each sample well of the final 96- or 384-well sample plate). Protocol A 25 µl reactions (dispensed to make four 5 µl reactions in a 384-well final sample plate) 4.A Dispense 15 µl of each PCR premix to a 96-well microtiter plate to generate a layout as shown in Figure 5 below. Figure 5: PCR Premix Plate Example of 96-well microtiter plate layout containing 15 µl of PCR premix per well, for 16 cdna samples dispensed 6 times. Each unique cdna sample is designated by a number and color-coded accordingly, to match the colors in Figure 4. 5.A Transfer 10 µl of the competitor mix from the competitor plate (prepared in Step 1) to the corresponding wells of the PCR premix plate (prepared in Step 4.A). See Figure 6. Mix and centrifuge the plate. 6.A Distribute 5 µl from the competitor mix + PCR premix plate (created in step 5.A) into 4 replicate wells of a new 384-well plate. See Figure 6. (After completing these steps, approximately 5 µl of the 25 µl PCR cocktail will be left over.) This 384-well sample plate will now have the same layout as shown in Figure 4B on page 13. MassARRAY QGE-iPLEX Application Guide 16 Doc R2.0, CO

21 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE. Competitor Mix Plate A 2.5x x x x x x10-13 PCR Premix Plate Final Sample Plate Figure 6: Protocol A (25 µl reactions dispensed to 5 µl reactions) Adding 10 µl competitor mix to each well of the PCR premix plate (containing 15 µl PCR premix) to create 25 µl reactions, then dispensing 5 µl of each final reaction cocktail to four replicate wells in the final sample plate. 7.A Seal and centrifuge the 384-well sample plate at 1000 RPM for 1 minute, and then proceed to Thermocyling rcpcr Reactions (page 19). Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

22 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE Protocol B 5 µl reactions 4.B Dispense 3 µl per reaction of the PCR premix to a new 384-well microtiter plate (for four replicates per reaction) or a new 96-well microtiter plate (for one replicate per reaction). 5.B Transfer 2 µl of the competitor mix from the competitor plate (prepared in step 1) to the corresponding wells of the PCR premix plate (prepared in step 4.B). See Figure 7 (384-well plate) or Figure 8 (96-well plate). This sample plate will now have the same layout as shown in Figure 4A (96-well plate) or Figure 4B (384-well plate) on page 13. Competitor Mix Plate A 2.5x x x x x x10-13 PCR Premix Plate Becomes Final Sample Plate Figure 7: Protocol B (5 µl reactions; 384-well plate) Adding 2 µl competitor mix to each well of the PCR premix plate, to create a final 384-well sample plate with 5 µl reactions. MassARRAY QGE-iPLEX Application Guide 18 Doc R2.0, CO

23 Chapter 3: Processing QGE-iPLEX Reactions 3.2 Real Competitive PCR for QGE Competitor Mix Plate A 2.5x x x x x x A 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 B 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 C 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 D 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 E 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 F 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 G 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 H 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 1E-18 1E-17 1E-16 1E-15 1E-14 1E-13 PCR Premix Plate Becomes Final Sample Plate Figure 8: Protocol B (5 µl reactions; 96-well plate) Adding 2 µl competitor mix to each well of the PCR premix plate, to create a final 96-well sample plate with 5 µl reactions. 6.B Seal the final sample plate and mix. Centrifuge the sample plate at 1000 RPM for 1 minute, and then proceed to Thermocyling rcpcr Reaction. Thermocycling rcpcr Reactions Thermocycle the final sample plate as follows: 94 C for 2 minutes 94 C for 30 seconds 56 C for 30 seconds 45 cycles 72 C for 1 minute 72 C for 5 minutes 4 C forever NOTE If not proceeding directly to the next step, the samples may be stored. Seal the sample plate(s), and store at -20º C. Do not store for more than 2 weeks. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

24 Chapter 3: Processing QGE-iPLEX Reactions 3.3 Neutralizing Unincorporated dntps (SAP Reaction) 3.3 Neutralizing Unincorporated dntps (SAP Reaction) NOTE If the sample plate(s) were frozen and stored after the previous step, let them thaw to room temperature before proceeding. NOTE The term sample plates (or wells) refers to your amplification product, prepared in Section Prepare the SAP enzyme solution as described in the following table. Table 6. SAP Enzyme Solution Reagent Water (HPLC grade) SAP Buffer (10x) SAP enzyme (1.7 U/μL) 2. Vortex the tube of SAP enzyme solution lightly and centrifuge. 3. Dispense 2 µl of the SAP enzyme solution into each sample well. For an automated process, see Appendix B Automated Liquid Handling Procedures, Section B Gently mix or vortex the sample plates, and centrifuge at 1000 RPM before incubating. 5. Incubate the sample plate(s) as follows: 1. 37º C for 40 minutes º C for 5 minutes. 3. 4º C forever. Volume (1rxn)* 1.53 μl 0.17 μl 0.30 μl Total Volume 2.00 μl *For more than one reaction, an overhang should be added to the volumes. See Section 3.1 on page While the sample plate(s) are incubating, begin preparing the QGE-iPLEX reaction cocktail. NOTE If not proceeding directly to the next step, the samples may be stored. Seal the sample plate(s), and store at -20º C. Do not store for more than 2 weeks. MassARRAY QGE-iPLEX Application Guide 20 Doc R2.0, CO

25 Chapter 3: Processing QGE-iPLEX Reactions 3.4 Creating the QGE-iPLEX Reaction (Extend Reaction) 3.4 Creating the QGE-iPLEX Reaction (Extend Reaction) In this section we will create the actual QGE-iPLEX product, via extension. In order to do this, it is important to adjust the extension primers. NOTE It is advised (but not required) that users reduce plex levels for quantitative reactions such as QGE, to minimize noise within a spectra which can compromise accuracy, reproducibility, and sensitivity. An upper limit of 24 plex is suggested. NOTE If the sample plate(s) were frozen and stored after the previous step, let them thaw to room temperature before proceeding. Adjusting the Extension Primers When conducting multiplexing experiments, adjusting the concentrations of oligos to equilibrate signal-to-noise ratios is required. The cocktail mix varies somewhat depending on whether a low plex or high plex reaction is being processed.! IMPORTANT Adjusting the primer mix is critical to successful multiplexing and requires the use of a SpectroCHIP array. If the extend primer concentration is not adjusted, an assay with a very low intensity primer peak will systematically fail when applied to samples as part of a multiplex. The minimum recommended method for adjusting extension primers for low plex (2 plex - 18 plex) reactions is a 2-tier method, dividing the primers into a low mass group and a high mass group. All primers in the high mass group are doubled in concentration with respect to the low mass group. For example, in a 18-plex, the 9 lowest mass primers would be at a concentration of 0.73 µm and the 9 highest mass primers would be at 1.46 µm in the final 9 µl reaction. The minimum recommended method for adjusting high plex (19 plex plex) extension primers is a 3-tier method, dividing the primers into a low mass group, a medium mass group, and a high mass group. All primers are adjusted in a three tier fashion, one tier for every twelve primers. For example, in a 36-plex, the 12 lowest mass primers would be at a concentration of 0.52 µm, the 12 middle mass primers would be at a concentration of 1.04 µm and the 12 highest mass primers would be at 1.57 µm in the final 9 µl reaction. Table 7. Primer Concentrations for Uniplex, Low Plex, and High Plex Reactions Uniplex 2-18 plex plex Low Mass 1.00 μm 0.73 μm 0.52 μm Medium Mass n/a n/a 1.04 μm High Mass n/a 1.46 μm 1.57 μm Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

26 Chapter 3: Processing QGE-iPLEX Reactions 3.4 Creating the QGE-iPLEX Reaction (Extend Reaction) More detailed protocols for extension primer adjustment are described in Appendix A Extention Primer Adjustment Methods. See also the Extension Primer Adjustment section in the QGE Software User s Guide on the QGE software disc (part # 11498). Preparing the QGE-iPLEX Reaction Cocktail 1. Prepare the QGE-iPLEX reaction cocktail using one of the following tables.! IMPORTANT Use Table 8 for a low plex (2 plex - 18 plex) reaction OR use Table 9 for a high plex (19 plex plex) reaction. Table 8. Low plex (2 plex - 18 plex) QGE-iPLEX reaction cocktail (same multiplexed assays, different DNAs) Reagent Conc. in 9 μl Volume (1rxn)* Water (HPLC grade) NA μl iplex Buffer Plus (10x) 0.222X μl iplex Termination mix 0.5x μl Primer mix (7 μm: 14 μm)** 0.73 μm: 1.46 μm μl iplex enzyme 0.5x μl Total Volume: μl *For more than one reaction, an overhang should be added to the volumes. See Section 3.1 on page 9. **The minimum recommended primer adjustment for low plex reactions is a 2-tier grouping. See Appendix A Extention Primer Adjustment Methods for more information. Note: If primer extension rate appears low for 25% or more of your assays increase termination mix and termination enzyme to 1X concentration. MassARRAY QGE-iPLEX Application Guide 22 Doc R2.0, CO

27 Chapter 3: Processing QGE-iPLEX Reactions 3.4 Creating the QGE-iPLEX Reaction (Extend Reaction) Table 9. High plex (19 plex plex) QGE-iPLEX reaction cocktail (same multiplexed assays, different DNAs) Reagent Conc. in 9 μl Volume (1rxn)* Water (HPLC grade) NA μl iplex Buffer Plus (10x) 0.222X μl iplex Termination mix 1x μl Primer mix (5 μm: 10 μm: 15 μm)** 0.52 μm:1.04 μm: 1.57 μm μl iplex enzyme 1x μl Total Volume: μl *For more than one reaction, an overhang should be added to the volumes. See Section 3.1 on page 9. **The minimum recommended primer adjustment for high plex reactions is a 3-tier grouping. See Appendix A Extention Primer Adjustment Methods for more information. 2. Vortex the tube of QGE-iPLEX reaction cocktail lightly. 3. Centrifuge the tube of QGE-iPLEX reaction cocktail at 1000 RPM for one minute. 4. Add 2 µl of QGE-iPLEX reaction cocktail to each sample well. For an automated liquid handling process, see Appendix B Automated Liquid Handling Procedures, Section B Gently mix or vortex the sample plates, and centrifuge at 1000 RPM before thermocycling. Thermocycling the QGE-iPLEX Reaction Cocktail Thermocycle the samples as follows: 94º C for 30 seconds 94º C for 5 seconds 52º C for 5 seconds 80º C for 5 seconds 72º C for 3 minutes 4º C forever 5 cycles 40 cycles For example, the ABI 9700 Thermocycler would be programmed as follows: 1 Hld 11 Tmp 40 Cycles 2 Hld 94 C 94 C 52 C 80 C 52 C 80 C 52 C 80 C 52 C 80 C 52 C 80 C 72 C 4 C 0:30 0:05 0:05 0:05 0:05 0:05 0:05 0:05 0:05 0:05 0:05 0:05 3:00? Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

28 Chapter 3: Processing QGE-iPLEX Reactions 3.5 Conditioning the QGE-iPLEX Reaction Products NOTE If not proceeding directly to the next step, the samples may be stored. Seal the sample plates, and store at -20º C. Do not store for more than 2 weeks. 3.5 Conditioning the QGE-iPLEX Reaction Products NOTE If the sample plates were frozen and stored after the previous step, let them thaw to room temperature before proceeding.! IMPORTANT Perform these steps on a clean plastic sheet. The excess resin that is scraped off the dimple plate will fall to the plastic sheet. The excess resin can be returned to its container for future use. When not in use, keep the container of Clean Resin tightly closed. This is necessary to prevent the drying out of the resin.! IMPORTANT Use the correct format for your sample set up: For 96 samples, use 15 mg resin. For 384 samples, use 6 mg resin. This conditioning step is crucial to optimize mass spectrometry analysis of the QGEiPLEX reaction products. 1. Using the elongated spoon, transfer resin from its container onto the dimple plate. 2. Use the scraper to spread resin into the wells of the dimple plate. Sweep the scraper from side to side across the dimple plate to spread the resin. Scraper Note: Make sure there is resin in each well. Resin MassARRAY QGE-iPLEX Application Guide 24 Doc R2.0, CO

29 Chapter 3: Processing QGE-iPLEX Reactions 3.5 Conditioning the QGE-iPLEX Reaction Products 3. Scrape excess resin off the dimple plate using the scraper. Scrape away from the small, rounded post. Return the excess resin to its container. 4. Let the resin stand in the dimple plate for at least 20 minutes. NOTE: For OncoCarta assays, let the resin stand in the dimple plate for only 10 minutes. 5. While letting the resin stand in the dimple plate, add water to the sample plate. For an automated process, see Appendix B Automated Liquid Handling Procedures, Section B.4. For 96 samples, add 41 µl nanopure water to each sample. For 384 samples, add 16 µl nanopure water to each sample. 6. Gently place the sample plate, upside-down, onto the dimple plate. 384-well sample microtiter plate (upside-down). Make sure the sample plate rests against this small, rounded post on the dimple plate; this aligns the wells in the sample plate with the wells in the dimple plate. Dimple plate 7. Holding the sample plate and the dimple plate together, gently flip them over so the resin falls out of the dimple plate into the wells of the sample plate. 384-well sample plate and dimple plate. Flip them over so the dimple plate is on top. Tap the dimple plate so the resin falls out into the sample plate. Make sure all the resin in the dimple plate falls out into the sample plate wells. If there are air bubbles in the wells, centrifuge the plate briefly. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

30 Chapter 3: Processing QGE-iPLEX Reactions 3.6 Dispensing onto SpectroCHIP Arrays 8. Rotate the sample plate on a rotator for five minutes, at room temperature. The rotator must rotate the sample plate 360º about the long axis. 9. Centrifuge the sample plate at 3200 g for five minutes. Processing of the QGE- iplex Reaction is now complete. NOTE If not proceeding directly to the next step, the samples may be stored. Seal the sample plates, and store at -20º C. Do not store for more than 2 weeks. 3.6 Dispensing onto SpectroCHIP Arrays NOTE If the sample plates were frozen and stored after the previous step, let them thaw to room temperature and then rotate and centrifuge (at 3200 g for five minutes) before transferring the reaction products to a SpectroCHIP array. After the QGE-iPLEX Reaction is complete, perform nanodispensing of QGE-iPLEX reaction products onto a SpectroCHIP array. For automatic dispensing instructions for 96 or 384 format, see the MassARRAY Nanodispenser User s Guide RS 1000 (Part #11558). 3.7 Defining Assays and Plates Once dispensing is complete, define how assays and plates are to be set up in the MassARRAY database. For instructions, see the Defining Assays and Defining Plates chapters in the QGE Software Guide (part # 11498). 3.8 Acquiring and Analyzing Spectra After assays and plates have been defined, acquire spectra using the MassARRAY mass spectrometer and analyze using QGE Analyzer Software. For instructions, see the MassARRAY Analyzer Compact User's Guide (Part # 11533). See also the QGE Software Guide (part # 11498). MassARRAY QGE-iPLEX Application Guide 26 Doc R2.0, CO

31 Chapter 3: Processing QGE-iPLEX Reactions 3.9 Normalizing Data 3.9 Normalizing Data Normalizing the data is recommended, to account for experimental variances, such as the amount of starting material and enzymatic efficiencies. Normalization can be performed using multiplex gene panels in conjunction with the normalization procedure developed by Vandesompele et. al. 8 See the application note Data Normalization Using Multiplexed Gene Panels for Quantitative Gene Expression Analysis with MassARRAY 3 available from the Sequenom web site. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

32 Chapter 3: Processing QGE-iPLEX Reactions 3.9 Normalizing Data NOTES: MassARRAY QGE-iPLEX Application Guide 28 Doc R2.0, CO

33 Appendix A Extension Primer Adjustment Methods Due to the inverse relationship between peak intensity and analyte mass, extend primers in QGE-iPLEX assays must be adjusted by concentration. The highest mass primer (~8500 Da) has a peak intensity 25% less than the average of the lower mass primers. Because of this, analyte signal-to-noise ratio estimation throughout the spectrum can pose a significant challenge to the Caller software. In the context of a genotyping reaction, analyte peaks can be missed, leading to genotyping errors. In addition to the inverse relationship between analyte mass and signal-to-noise ratio, nonpredictable variations in peak heights can occur. These variations may stem from inconsistent oligonucleotide quality and poor desorption/ionization behavior in MALDI. Optical density measurements to assess oligonucleotide quality are not sufficient since fragments from partial synthesis and other absorbing contaminants contribute to the overestimation of full-length oligonucleotide concentration. Over-estimation of primer concentration prompts over-dilution, resulting in lower signal intensity in the spectrum and genotype calling problems. To overcome this situation, primer mixes must be tested in MALDI-TOF MS prior to genotyping. A mixture of extend primers containing each primer at the same concentration will be analyzed on the MassARRAY platform. Typer Analyzer Software will create a primer adjustment report that identifies primers to adjust within the mix and recommends a ratio for adjustment (see the Primer Adjustment Report section in Appendix B of the Typer User s Guide, part #11557). Given the inverse relationship between analyte mass and peak signal-to-noise ratio, the primer adjustment report specifies the high mass primers to be adjusted. To streamline the process flow, we describe four options for preparing QGE-iPLEX primer cocktails. The first three methods involve grouping the primers into 2, 3 or 4 tiers according to mass and adjusting the concentration. The fourth method (regression) involves mixing every primer at a specific concentration according to mass and equilibrating each primer in the spectrum using specific concentrations. SEQUENOM s field applications scientists will advise and train you on the best methods for your application. A general description of each of the primer adjustment methods follows. Option A: Two- Tier Method Option A involves doubling the concentration of high and low mass primers in two groups, as shown in Figure 9. Low Mass High Mass Figure 1: Two-Tier Primer Adjustment Method Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

34 Appendix A: Option B: Three-Tier Method Prepare the 7/14 µm QGE-iPLEX extend primer mix as follows: 1. Divide the extension primers into 2 groups, LOW mass, and HIGH mass. 2. Calculate the LOW mass group for 7 µm in the final QGE-iPLEX extend primer mix. 3. Calculate the HIGH mass group for 14 µm in the final QGE-iPLEX extend primer mix. 4. Calculate the remaining water volume. This is the easiest method to use for low plex reactions (2-18 plex). However, this may result in lower call rates for higher plex reactions. For maximal call rates for high plex reactions, use Option B, C, or D. Option B: Three-Tier Method Option B involves doubling the concentration of high and low mass primers in three groups, as shown in Figure 10. 1st Group 2nd Group 3rd Group Figure 2: Three-Tier Primer Adjustment (Option B) The first group of low mass primers are mixed at 5.0 µm, the medium mass group at 10 µm, and the high mass group at 15 µm. Prepare the 5/10/15µM QGE-iPLEX extend primer mix as follows: 1. Divide the extension primers into 3 groups, LOW mass, MEDIUM mass, and HIGH mass. 2. Calculate the LOW mass group for 5 µm in the final QGE-iPLEX extend primer mix. 3. Calculate the MEDIUM mass group for 10 µm in the final QGE-iPLEX extend primer mix. 4. Calculate the HIGH mass group for 15 µm in the final QGE-iPLEX extend primer mix. 5. Calculate the remaining water volume. MassARRAY QGE-iPLEX Application Guide 30 Doc R2.0, CO

35 Appendix A: Option C: Four-Tier Method Option C: Four- Tier Method Option C involves doubling the concentration of high and low mass primers in four groups, as shown in Figure 11. 1st Group 2nd Group 3rd Group 4th Group Figure 3: Four-Tier Primer Adjustment Method (Option C) The first group of low mass primers are mixed at 7.0 µm, the following group at 9.3 µm, the third group at 11.6 µm, and the higher mass group at 14.0 µm. Prepare the 7/9.3/11.6/ 14 µm QGE-iPLEX extend primer mix as follows: 1. Divide the extension primers into 4 groups from LOW mass to HIGH mass. 2. Calculate the LOW mass group for 7 µm in the final QGE-iPLEX extend primer mix. 3. Calculate the two MEDIUM mass groups for 9.3 and µm in the final QGEiPLEX extend primer mix. 4. Calculate the HIGH mass group for 14 µm in the final QGE-iPLEX extend primer mix. 5. Calculate the remaining water volume. Option D: Regression Method Option D involves mixing every primer at a specific concentration according to mass. Knowing the mass, it is possible to equilibrate each primer in the spectrum using specific concentrations. The following relation can be derived: Intensity = -301Ln(Mass) This relation does not consider oligonucleotide purity and non-predictable sequencespecific desorption/ionization variance; however, it is still useful in providing a good starting point. A sample plot is shown in Figure 12. Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

36 Appendix A: Option D: Regression Method Intensity Mass (Da) Figure 4: Relation of Peak Intensity and Mass A Microsoft Excel spreadsheet can be created to calculate concentration and dilution to apply to each primer within a reaction. For example, apply the following formula to calculate the diluted oligo concentration (found in column F when exported to Excel from the Extension Primer Adjustment table found in the PlateEditor): (LN(Mass)-7.82) * oligo stock concentration = target primer concentration in mixture Knowing the initial concentration and volume, additional water volume (µl) can be calculated (column H), and an equivalent volume of diluted primers can be used to create the working mix. Follow the instructions in Figure 5 to prepare the extend primer mix using the linear adjustment method. Figure 5: Preparing the extend primer mix (linear adjustment method) MassARRAY QGE-iPLEX Application Guide 32 Doc R2.0, CO

37 Appendix A: Option D: Regression Method Figure 6A shows a 23-plex equimolar iplex primer mix before adjustment. The last seven oligos, and particularly the one at ~7700 Da, have intensities close to detection limits. An iplex reaction on a heterozygous sample with below average yields would produce allelic peaks below detection thresholds. Figure 6B shows the same primers mixed using the regression method (Option D). A. Non-adjusted Equimolar Primer Mix B. Adjusted with Regression Method Figure 6: Comparison of Non-adjusted 23-plex Primer Mix Versus Adjusted Mix Using Option D, Regression Method Doc R2.0, CO MassARRAY QGE-iPLEX Application Guide

38 Appendix A: Option D: Regression Method Notes: MassARRAY QGE-iPLEX Application Guide 34 Doc R2.0, CO

39 Appendix B Automated Liquid Handling Procedures Source Plate The microtiter plate from which liquid is aspirated. Destination Plate The microtiter plate to which liquid is dispensed. This appendix covers processing of QGE-iPLEX reactions on the MassARRAY Matrix Liquid Handler, dispensing the contents of one source plate to either one or two 384-well destination plates. The two-plate option requires you to prepare increased reagent volumes and to select two-plate method options in the Matrix software. See the MassARRAY Liquid Handler User s Guide (Part # 11541) for further details. NOTE The term sample plate(s) refers to the 384-well microtiter plate(s) of amplification products ( sample ) on which you want to perform the QGE-iPLEX reaction. B.1 Daily and Weekly Maintenance For complete instructions on maintenance tasks, see the MassARRAY Matrix Liquid Handler User s Guide (Part #11541). Maintenance methods are located in the Maintenance Methods folder within the Matrix ControlMate software. NOTE When opening a method, choose Cancel in the File Locked dialog box. Daily Tasks Each day, before using the liquid handler to process an QGE-iPLEX reaction: 1. Check the wash system tanks. Fill the supply tank and empty the waste tank if necessary. 2. Make sure the wash station is on deck position Run the Liquid Handler Startup Routine method. 4. At the end of each day, run the Liquid Handler Shutdown Routine method. Weekly Tasks If instrument is in daily use, this maintenance can be performed bimonthly instead of weekly. At the end of each week, after processing of reactions is completed and the Liquid Handler Shutdown Routine method has been run: 1. Run the Liquid Handler Weekly Maintenance Routine method. 2. Wipe down the instrument, deck, and work area with water or mild detergent. Doc R1.0, CO MassARRAY QGE-iPLEX Application Guide June 19, 2009

40 Appendix B: Automated Liquid Handling Procedures B.2 Adding the SAP Enzyme Solution B.2 Adding the SAP Enzyme Solution 1. Perform the PCR reactions as described in Section 3.2 Real Competitive PCR for QGE on page Prepare the SAP enzyme solution as described in Section 3.3 Neutralizing Unincorporated dntps (SAP Reaction) on page Obtain a new 96-well microtiter plate (Sarstedt Vee Bottom). If using one destination plate, pipette 10 µl of the SAP enzyme solution into each well in rows A-G. If using two destination plates, pipette 20 µl of the SAP enzyme solution into each well in rows A-G. NOTE The SAP enzyme solution is moderately viscous. Use care when pipetting to minimize loss of solution due to adhesion. Be sure to pipette into the centers of microtiter plate wells droplets must not be placed so they adhere to well-walls. Also, make sure there are no air bubbles in the wells. Centrifuge a microtiter plate at 1600 RPM for one minute to remove air bubbles and collect liquid at the center of wells. NOTE The 38% recommended overhang can be reduced to 30% when a single pipette repeater is used to fill the 96-well Vee Bottom plate. 4. Place the 96-well plate of the SAP solution on position 1 of the liquid-handler deck. 1 2 SAP Wash Station Diagram of the liquid-handler deck with 96-well SAP plate added to position Centrifuge the 384-well sample plate(s) containing amplification products at 1000 RPM for 1 minute. 6. If the 384-well sample plate(s) are sealed, remove the plate seal. MassARRAY QGE-iPLEX Application Guide 36 Doc R1.0, CO June 19, 2009

41 Appendix B: Automated Liquid Handling Procedures B.2 Adding the SAP Enzyme Solution 7. Place the 384-well sample plate(s) on plate flattener(s). Then, place one sample plate on position 3 of the liquid-handler deck. If there is a second sample plate, place it on position SAP Wash Station Diagram of the liquid-handler deck with 384-well sample plate added to position 3. Sample 384-well plates must be on plate flatteners 3 4! CAUTION If the amplification products must be transferred from the original plate to another plate, you must transfer them to a plate that has been previously cycledin the thermocycler. These cycled plates will fit appropriately on the plate flatners of the matrix whereas noncycled plates may fit too loosely. 8. On the liquid handler controller PC, open the 384 Methods folder and the iplexhme Methods folder. Then, run the SAP Addition (96 to 384) method. 9. When prompted for How Many 384 Plates, leave the default setting at 1 for one destination plate, or change to 2 for two destination plates. Edit Global Values dialog box If you are dispensing to 2 sample plates, you must type 2 in the How Many 384 Plates box. 2 µl of SAP enzyme solution is added to each well in the 384-well sample plate(s). Doc R1.0, CO MassARRAY QGE-iPLEX Application Guide June 19, 2009