Sample & Assay Technologies PyroMark® Q24 Validation
Contents
1. Table 2 Plate setup for bias and repeatability determination 1 2 3 4 5 6 7 8 A C A B B C C A B B A e A B B C A B C C A B A C B C A 3 Save the Run Setups to a USB memory stick supplied with the PyroMark Q24 System 4 Printa list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup for each Run Setup Select Pre Run Information from the Tools menu and when the report appears click 3 12 PyroMark Q24 Validation Oligo Handbook 09 2009 Protocol 3 Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution Series Important points before starting EB Accurate pipetting is critical to obtain the correct mixtures The method described below involves successive mixing of equal volumes of solutions This reduces errors in pipetting It is still critical that the same pipetting technique is used for all mixes to ensure that equal volumes are indeed dispensed M The dilution buffer supplied with the PyroMark Q24 Validation Oligo contains an agent that effectively eliminates adsorption of the oligonucleotides to plastic surfaces that might adversely affect performance It is important that this buffer is used where specified The PyroMark Q24 Validation Oligo is itself stored in this buffer Procedure 1 The dilution buffer provided with the PyroMark Q24 Validation Oligo needs to be diluted before use Prepare 1x dilution buffer by mixing 600 ul of 10x dilution2. D 1 27 5 31 2 2 27 5 29 89 3 27 5 29 89 C 1 50 51 88 2 50 52 62 3 50 52 27 F 72 5 74 76 2 72 5 74 66 3 72 5 75 31 G 83 8 85 28 2 83 8 85 53 3 83 8 85 68 B 95 95 30 2 95 95 40 3 95 95 73 These values are given as an example only Actual values must be determined e E EE 24 PyroMark Q24 Validation Oligo Handbook 09 2009 Linearity Plot Linear fit 1 905 0 9967x E Polynomial fit 5 0 4493 1 097x 0 001002x lt 0 20 40 60 80 100 Expected B Difference Plot Allow able non linearity 3 e x 85 e Se 5 ae E o 0 20 40 60 80 100 Concentration Figure 6 Example of linearity analysis N The linear fit and the polynomial fit are shown graphically The polynomial fit is statistically significant The difference plot shows that the data is well within the limits of allowed nonlinearity of 3 percentage units PyroMark Q24 Validation Oligo Handbook 09 2009 25 Protocol 6 Analysis of Bias and Repeatability Claimed performance for both CpG and AQ modes For 0 5 2 picomoles of the PyroMark Q24 Validation Oligo using the method described here the method has been demonstrated to give the following performance E Repeatability measured as standard deviation for 8 replicates better than 3 percentage units in the range 596 C to 9596 C EB Bias less than 5 percentage units for a mean of 8 replicates in the range 596 C to 9596
3. Poor linearity Pipetting errors Make sure to carefully follow the instructions for diluting the PyroMark Q24 Validation Oligo in Protocol 3 Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution Series To ensure comparable dilutions we strongly recommend to pipet aliquots of the same volume without changing any settings on the pipet between mixes Inverted slope in linearity test 30 596 and 9596 C mixes Make sure to label tubes clearly and not mix up exchanged tubes during dilution of the PyroMark Q24 Validation Oligo PyroMark Q24 Validation Oligo Handbook 09 2009 Appendix A Preparing the PyroMark Q24 Vacuum Workstation This protocol is a description of how to prepare the PyroMark Q24 Vacuum Workstation before using it with the PyroMark Q24 Validation Oligo Procedure 1 Fill 5 separate troughs supplied with the PyroMark Q24 Vacuum Workstation as follows m Approximately 50 ml ethanol 70 1 m Approximately 40 ml PyroMark Denaturation Solution 2 m Approximately 50 ml PyroMark Wash Buffer 3 Approximately 50 ml high purity water 4 Approximately 70 ml high purity water 5 A suggested setup is shown in Figure 8 Refill the troughs to these levels whenever necessary Figure 8 Positions on the PyroMark Q24 Vacuum Workstation 2 Switch on the vacuum pump so Apply vacuum to the tool by opening the vacuum switch 4 Wash the filter probes by lowering the pro
4. PyroMark PCR Kit For 200 reactions 2x PyroMark PCR 978703 200 Master Mix includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl and dNTPs 10x CoralLoad Concentrate 5x Q Solution 25 mM MgCl and RNase Free Water EpiTect Bisulfite Kit 48 EpiTect Bisulfite Spin Columns 59104 48 Reaction Mix DNA Protect Buffer Carrier RNA Buffers EpiTect PCR Control Human control DNA set containing 59695 DNA Set 100 both bisulfite converted methylated and unmethylated DNA and unconverted unmethylated DNA for 100 control PCRs Other kit sizes formats available see www qiagen com For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor EEE s SE E SC E ZY R RB BP PPPPRPRPRPRPRFRFPFPEBB SR PyroMark Q24 Validation Oligo Handbook 09 2009 35 Notes 36 PyroMark Q24 Validation Oligo Handbook 09 2009 Notes PyroMark Q24 Validation Oligo Handbook 09 2009 37 Notes 38 PyroMark Q24 Validation Oligo Handbook 09 2009 Trademarks QIAGEN CoralLoad PyroMark Pyrosequencing Pyrogram Q Solution QIAGEN Group Microsoft Microsoft Corporation Milli Q Millipore Corporation Sepharose GE Healthcare Limited License Agreement Use of this prod
5. 09 2009 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier For use on the PyroMark Q24 E PyroMark Q24 cat no 9001514 M PyroMark Q24 Software cat no 9019062 E PyroMark Q24 Plate cat no 979201 E PyroMark Q24 Cartridge cat no 979202 BE PyroMark Q24 Vacuum Workstation cat no 9001518 220V 9001516 110V 9001519 100V BE PyroMark Gold Q24 Reagents cat no 970802 M PyroMark Binding Buffer cat no 979006 E PPyroMark Denaturation Solution cat no 979007 M PyroMark Wash Buffer concentrate cat no 979008 M PyroMark Annealing Buffer cat no 979009 E Streptavidin Sepharose High Performance GE Healthcare cat no 17 5113 01 www gelifesciences com M Plate mixer for immobilization to beads M Heating block capable of attaining 80 C M 24 well PCR plate or strips E Strip caps BE 1 5 ml or 2 ml microcentrifuge tubes for dilution of the PyroMark Q24 Validation Oligo E Permanent pen for labeling tubes E High purity water Milli Q 18 2 MQ x cm or equivalent M Ethanol 70 mB Pipets adjustable E Sterile pipet tips Ensure that instruments have been checked and calibrated according to the manufacturer s recommendations PyroMark Q24 Validation Oligo Handbook 09
6. 2009 9 Protocol 1 Setting Up a PyroMark Q24 Validation Oligo Assay Important point before starting mB For further information on how to create an Assay Setup and a Run Setup see the PyroMark Q24 Software User Guide E The Assay can be set up using either AQ or CpG mode Procedure 1 Set up an assay for the PyroMark Q24 Validation Oligo by using the PyroMark Q24 Software 2 Click in the toolbar and select New AQ Assay or New CpG Assay 3 Type the following sequence in Sequence to Analyze TAYGGTITGA For more information on how to create an Assay Setup file see the PyroMark Q24 Software User Guide 4 Click the Generate Dispensation Order icon to get the following nucleotide dispensation order AQ CTGACTGTG CpG ATGATCGTG C T G A C T G T G 5 Figure 2 Histogram for AQ mode The first and third nucleotide additions are blank dispensations and serve as negative controls The fifth and sixth dispensations constitute the variable position created by the mixing of the 2 oligonucleotides 5 Click H in the toolbar to save the assay ES SEEEEBRSSS E EEEOEE E RRE RRRRRR RRS RR GBSBS ETZTTEEBBSSEE E PR SE ER 10 PyroMark Q24 Validation Oligo Handbook 09 2009 Protocol 2 Run Setup for Performance Test of the PyroMark Q24 System Important points before starting BE For instructions on how to create a new Run Setup see the PyroMark Q24 Software User Guide E It is recommended to set up
7. C The mixes A B and C with 596 C 9596 C and 5096 C respectively are used to determine repeatability bias and intermediate precision Procedure 1 Open the run files for BiasRepeatability 0 5picomol and BiasRepeatability 2picomol in the PyroMark Q24 Software and analyze all wells All wells should be given Passed quality shown as a blue bar in the lower field of the well and with C indicated in a blue rectangle in the Pyrogram output 5196 T 4996 250 200 150 100 0 E S C T G A C T G T G 5 Figure 7 Example of AQ assay result from a 50 mix tube C0 1 2 Determine the single peak heights Ideally the peaks should be between 30 10 RLU for samples with 0 5 picomoles of template and above 120 40 RLU for samples with 2 picomoles of template To obtain peak height values select Export Peak Heights from the Tools menu Save the data in a suitable format csv or tsv Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well as described below 26 PyroMark Q24 Validation Oligo Handbook 09 2009 3 Select the AQ CpG Analysis Results from the Reports menu to open the analysis result report 4 Save the data in a suitable format csv or tsv 5 Open the data file in the software 6 Prepare a table with expected and actual values An example is shown in Table 7 on page 24 7 The data obtained fr
8. 4 Validation Oligo Handbook 09 2009 Introduction The PyroMark Q24 Validation Oligo provides a means to check the performance of the PyroMark Q24 system Principle and procedure The product consists of 2 biotinylated oligonucleotides that differ in sequence in one position synthesized as A or G A variable position is generated by mixing the 2 oligonucleotides in different proportions C or T is incorporated upon sequencing and the variable position is analyzed as C Replicates of the mixes are used to determine linearity bias and repeatability These determinations constitute the performance test of the system The limits of the proportions of the 2 mixes 596 and 9596 have been carefully chosen to coincide with the generally accepted limits for reliable quantification as determined by in house evaluation and published data 2 8 The performance test is valid for the whole PyroMark Q24 system since the mixes are prepared through PyroMark Q24 Vacuum Workstation before analysis in the PyroMark Q24 instrument Both oligonucleotides can form an internal stem loop structure This structure enables self priming of the oligonucleotides for extension by the DNA polymerase and eliminates the need for a sequencing primer in the Pyrosequencing reaction Figure 1 shows the structure of the oligonucleotides n 3 3 cu EV x AB R Lo ATACCAAAC B aun M sua TCC CAR AC Py dea e B Sequenced region O Nr TAYGGTITG oe J ATRCCAA
9. 9 2009 Series in triplicates to Plate 1 in the same pattern as in the Run Setup for Linearity_0 5picomol see the Pre Run Information report from Protocol 2 Run Setup for Performance Test of the PyroMark Q24 System The 3 wells that remain can be used as negative controls Add 20 ul 1x dilution buffer instead of oligonucleotides The total volume per well should be 80 ul after addition of the PyroMark Q24 Validation Oligo mixes 5 Plate 2 Pipet 20 pl of each PyroMark Q24 Validation Oligo mix 0 1 uM tubes AO 1 through GO 1 from Protocol 3 Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution Series in triplicates to Plate 2 in the same pattern as in the Run Setup for Linearity 2picomol see the Pre Run Information report from Protocol 2 Run Setup for Performance Test of the PyroMark Q24 System The 3 wells that remain can be used as negative controls Add 20 ul 1x dilution buffer instead of oligonucleotides The total volume per well should be 80 ul after addition of the PyroMark Q24 Validation Oligo mixes 6 Plate 3 Pipet 20 pl of the first 3 PyroMark Q24 Validation Oligo mixes 0 025 uM tubes A0 025 through C0 025 from Protocol 3 Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution Series in replicates of eight to Plate 3 in the same pattern as in the Run Setup for BiasRepeatability 0 5picomol see the Pre Run Information report from Protocol 2 Run Setup for Perform
10. AC O gt Biotin Figure1 Structure of the PyroMark Q24 Validation Oligos JN The open structure of the oligonucleotides The self primed structure of the oligonucleotides with the analyzed sequence indicated The terminology for performance parameters are definitions adapted from reference 1 see References page 33 Linearity Ability within a given measuring interval to provide measurement results that are directly proportional to the value of 96C in the sample Bias Difference between the results of measurement and a true value of C Reproducibility Precision of successive measurement results for C carried out under essentially unchanged conditions of measurement for example replicates PyroMark Q24 Validation Oligo Handbook 09 2009 7 Description of protocols The workflow below illustrates the assay procedure Workflow of PyroMark Q24 Validation Oligo procedure Assay and Run Setup Sample preparation Dilution of each PyroMark Q24 Assay Setup Protocol 1 Validation Oligo Protocol 3 Y y Preparation of mixes with different C Protocol 3 Y Dilution of mixes to 0 1 uM and Run Setup Protocol 2 0 025 uM Protocol 3 Y Preparation of samples Protocol 4 Y Y Immobilization Protocol 4 lc c ud PyroMark Q24 run Protocol 4 Y Analysis of PyroMark Q24 run for linearity Protocol 5 Y Analysis of PyroMark Q24 run for bias and repeatability Protocol 6 Y Report 8 PyroMark Q24 Validation Oligo Handbook
11. Appendix B Emptying the Waste Container and Troughs 32 References 33 Ordering Information 34 PyroMark Q24 Validation Oligo Handbook 09 2009 3 Kit Contents PyroMark Q24 Validation Oligo Catalog no 979204 Number of assays 3 PyroMark Q24 Validation Oligo 5 20 uM 70 ul PyroMark Q24 Validation Oligo 9596 20 uM 70 ul Dilution Buffer 2x 1 7 ml Handbook 1 Storage The PyroMark Q24 Validation Oligo should be stored at 20 C upon arrival Repeated thawing and freezing 74 x should be avoided The PyroMark Q24 Validation Oligo is stable until the expiration date when stored under these conditions Product Use Limitations The PyroMark Q24 Validation Oligo is intended for molecular biology applications This product is neither intended for the diagnosis prevention or treatment of a disease nor has it been validated for such use either alone or in combination with other products Therefore the performance characteristics of the products for clinical use i e diagnostic prognostic therapeutic or blood banking are unknown All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product litera
12. Linearity Bias and Repeatability Things to do before starting Bm Follow the instructions in PyroMark Q24 User Manual to install the PyroMark Q24 M Place the PyroMark Q24 Plate Holder on a heating block at 80 C for use in step 26 amp Allow all required reagents and solutions to reach room temperature 15 25 C before starting EB Label 4 PyroMark Q24 Plates as follows Plate 1 Plate 2 Plate 3 Plate 4 Procedure 1 Gently shake the bottle containing Streptavidin Sepharose High Performance until it is a homogeneous solution 2 Prepare a master mix for DNA immobilization according to Table 6 Prepare a volume at least 1096 greater than that required for the total number of reactions to be performed This protocol calls for 4 x 24 96 reactions Table 6 Master mix for DNA immobilization Number of samples 1 110 Streptavidin Sepharose High Performance oH id PyroMark Binding Buffer 40 ul 4 4 ml High purity water 18 ul 1 98 ml Total volume 60 pl 6 60 ml Provides a sufficient amount for the 4 x 24 96 samples required 3 Add 60 ul of the master mix to all 24 wells of four 24 well PCR plates Label the plates as follows Plate 1 Plate 2 Plate 3 Plate 4 4 Plate 1 Pipet 20 pl of each PyroMark Q24 Validation Oligo mix 0 025 pM tubes A0 025 through G0 025 from Protocol 3 Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution 16 PyroMark Q24 Validation Oligo Handbook 0
13. Remove the PyroMark Q24 Plates from the plate holder and let the samples cool to room temperature 15 25 C for at least 5 min PyroMark Q24 Validation Oligo Handbook 09 2009 19 28 29 30 Open the plate holding frame and place the PyroMark Q24 Plate 31 32 33 34 35 36 37 38 39 40 41 42 43 20 Note Since only 1 plate can be processed on the PyroMark Q24 at any given time keep the other plates on the bench at room temperature Load a PyroMark Q24 Cartridge with the appropriate volumes of PyroMark Gold Q24 Reagents as given in the Pre Run Information report for Linearity O 5picomol from Protocol 2 Run Setup for Performance Test of the PyroMark Q24 System The Pre Run Information report found in the Tools menu at run setup see the PyroMark Q24 Software User Guide provides information about the volume of nucleotides enzyme mixture and substrate mixture needed for the assay Open the cartridge gate and insert the filled PyroMark Q24 Cartridge with the label facing out Push the cartridge in fully and then push it down Ensure the cartridge is properly inserted and close the gate Plate 1 on the heating block Close the plate holding frame and the instrument lid Insert the USB memory stick containing the run file into the USB port at the front of the instrument Do not remove the USB stick before the run is finished Select Run in the main menu usin
14. September 2009 PyroMark Q24 Validation Oligo Handbook For performance check of the PyroMark Q24 system 89096 00000 QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 4 Product Use Limitations 4 Product Warranty and Satisfaction Guarantee 4 Technical Assistance 5 Quality Control 5 Safety Information 5 Introduction 7 Principle and procedure 7 Description of protocols 8 Equipment and Reagents to Be Supplied by User 9 Protocols E Setting Up a PyroMark Q24 Validation Oligo Assay 10 E Run Setup for Performance Test of the PyroMark Q24 System 11 EH Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution Series 13 E Determination of Linearity Bias and Repeatability 16 B Analysis of Linearity 22 E Analysis of Bias and Repeatability 26 Troubleshooting Guide 28 Appendix A Preparing the PyroMark Q24 Vacuum Workstation 31
15. ance Test of the PyroMark Q24 System The total volume per well should be 80 ul after addition of the PyroMark Q24 Validation Oligo mixes 7 Plate 4 Pipet 20 pl of the first 3 PyroMark Q24 Validation Oligo mixes 0 1 uM tubes AO 1 through CO 1 from Protocol 3 Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution Series in replicates of eight to Plate 4 in the same pattern as in the Run Setup for BiasRepeatability 2picomol see the Pre Run Information report from Protocol 2 Run Setup for Performance Test of the PyroMark Q24 System The total volume per well should be 80 ul after addition of the PyroMark Q24 Validation Oligo mixes 8 Seal the PCR plates Plate 1 through Plate 4 using strip caps PyroMark Q24 Validation Oligo Handbook 09 2009 17 9 Agitate Plate 1 at room temperature 15 25 C for 5 min at 1400 rpm Sepharose beads sediment quickly If more than 1 min has elapsed since the plate was agitated agitate again for 1 min before capturing the beads During this step prepare the PyroMark Q24 Vacuum Workstation for sample preparation see Appendix A page 31 10 Add 25 ul of PyroMark Annealing Buffer to each well of PyroMark Q24 Plate 1 Keep one of the PyroMark Q24 Plate Holders supplied with the PyroMark Q24 Vacuum Workstation at room temperature 15 25 C and use it as support when preparing and moving the plates Since the oligonucleotides are self primed no sequenci
16. and laws OSHA Occupational Safety and Health Administration United States of America t ACGIH American Conference of Government Industrial Hygienists United States of America COSHH control of Substances Hazardous to Health United Kingdom Be sure to observe federal state and local environmental regulations for the disposal of laboratory waste The following item is required mB High purity water Milli Q 18 2 MO x cm www millipore com or equivalent Procedure 1 Ensure that no vacuum is applied to the vacuum tool the vacuum switch is closed Off and the vacuum pump is switched off 2 Discard any solutions left in the troughs 3 Rinse the troughs with high purity water or replace them if necessary 4 Empty the waste container The cap can be removed without disconnecting the tubing 5 If the PyroMark Q24 Vacuum Workstation must be cleaned for dust or spillage follow the instructions in Cleaning the PyroMark Q24 Vacuum Workstation in the PyroMark Q24 User Manual 32 PyroMark Q24 Validation Oligo Handbook 09 2009 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at ww
17. bes into high purity water trough 5 Flush the probes with 70 ml high purity water Make sure that the water is being transferred to the waste container If it is not then make sure that the tubing is connected correctly and is not broken Broken tubing should be replaced see Replacing the tubing in the PyroMark Q24 User Manual 5 Make sure that the waste filter is dry If the filter is wet it should be replaced see Replacing the waste filter in the PyroMark Q24 User Manual 6 Close the vacuum switch on the tool Off and place the tool in the Parking P position 7 Refill trough 5 with 70 ml high purity water PyroMark Q24 Validation Oligo Handbook 09 2009 31 Appendix B Emptying the Waste Container and Troughs WARNING Hazardous chemicals The PyroMark Denaturation Solution used with the PyroMark Q24 Vacuum Workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations
18. buffer with 5400 pl of high purity water Note The agent may cause bubble formation during pipetting 2 Prepare 1 5 ml or 2 ml microcentrifuge tubes for the dilution series Label the tubes as follows Al B1 C1 DI EI F1 Gl AO 1 BO 1 CO 1 DO 1 EO 1 FO 1 GO 1 A0 025 B0 025 C0 025 DO 025 E0 025 F0 025 G0 025 3 Pipet 30 pl of the PyroMark Q24 Validation Oligo 5 20 uM into the tube marked A1 4 Pipet 30 pl of the PyroMark Q24 Validation Oligo 95 20 uM into the tube marked B1 5 Add 570 ul each of dilution buffer 1x from step 1 to tubes A1 and B1 to generate 1 uM solutions of each PyroMark Q24 Validation Oligo Mix by pipetting up and down To ensure comparable dilutions we strongly recommend to pipet the 30 ul aliquots and 570 ul without changing any settings on the pipet between mixes PyroMark Q24 Validation Oligo Handbook 09 2009 13 6 Prepare solutions for tubes C1 through G1 as shown in Table 3 Table 3 Preparation of PyroMark Q24 Validation Oligo mixes with different C contents Tube label Mix together Final volume C Al 600 ul 5 B1 600 ul 95 Cl 200 ul Al 200 ul B1 400 ul 50 D1 100 ul A1 100 ul C1 200 ul 27 596 E 100 ul A1 100 ul D1 200 ul 16 396 F1 100 ul B1 100 ul C1 200 ul 72 596 Gl 100 ul B1 100 ul F1 200 ul 83 896 7 Prepare solutions for tubes AO 1 through GO 1 by diluting each solution A1 through G1 to 0 1 uM as shown in Table 4 Tabl
19. ders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 CILLI USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN i Sample amp Assay Technologies
20. e 4 Dilution of the PyroMark Q24 Validation Oligo mixes for tubes A0 1 through GO 1 Component Volume Concentration Solutions A1 through G1 from 55 6 30 ul 1 uM 1x Dilution buffer 270 ul Solutions A0 1 through GO 1 300 pl 0 1 uM Make sure that the 10x dilution buffer supplied with the PyroMark Q24 Validation Oligo is diluted with high purity water before use See step 1 8 Prepare solutions for tubes A0 025 through G0 025 by performing a second dilution of each solution A0 1 through G0 1 to 0 025 uM as shown in Table 5 14 PyroMark Q24 Validation Oligo Handbook 09 2009 Table 5 Dilution of the PyroMark Q24 Validation Oligo mixes for tubes A0 025 through G0 025 Component Volume Concentration Solutions A0 1 through GO 1 from sob 60 ul 0 1 uM 1x Dilution buffer 180 ul Solutions A0 025 through G0 025 240 ul 0 025 uM Make sure that the 10x dilution buffer supplied with the PyroMark Q24 Validation Oligo is diluted with high purity water before use See step 1 The remaining volumes of PyroMark Q24 Validation Oligos in tubes A1 through G1 can be stored at 20 C for up to 1 month Repeated thawing and freezing 24 x should be avoided A PyroMark Q24 Validation Oligo Handbook 09 2009 15 Protocol 4 Determination of
21. g the 4 and screen buttons and press OK Select the run file Linearity 0 5picomol using the and screen buttons To view the contents of a folder select the folder and press Select To go back to the previous view press Back When the run file is selected press Select to start the run When the run is finished and the instrument confirms that the run file has been saved to the USB memory stick press Close Open the instrument lid Open the cartridge gate and remove the PyroMark Q24 Cartridge by lifting it up and pulling it out Close the gate Open the plate holding frame and remove the PyroMark Q24 Plate from the heating block Close the plate holding frame and the instrument lid Clean the PyroMark Q24 Cartridge if you choose not to immediately continue with the next plate see the PyroMark Gold Q24 Reagents Handbook PyroMark Q24 Validation Oligo Handbook 09 2009 44 Refill the PyroMark Q24 Cartridge with the appropriate volumes of PyroMark Gold Q24 Reagents as given in the Pre Run Information report for Linearity 2picomol from Protocol 2 Run Setup for Performance Test of the PyroMark Q24 System The Pre Run Information report found in the Tools menu at run setup see the PyroMark Q24 Software User Guide provides information about the volume of nucleotides enzyme mixture and substrate mixture needed for the assay 45 Open the cartridge gate and insert the filled Py
22. itate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of PyroMark Q24 Validation Oligo is tested against predetermined specifications to ensure consistent product quality Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component PyroMark Q24 Validation Oligo Handbook 09 2009 5 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 6 PyroMark Q2
23. l and no peaks in the Pyrogram Clean the PyroMark Q24 Cartridge and check that it is working properly Discard the PyroMark Q24 Cartridge according to federal state and local environmental regulations for disposal of laboratory waste 29 h Comments and suggestions Buffers or reagents Follow the instructions supplied with the incorrectly diluted or reagents incorrectly stored PyroMark Q24 Validation Follow the instruction in the protocols for Oligo not correctly preparing the PyroMark Q24 Validation Oligo prepared Make sure to dilute the PyroMark Q24 Validation Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water Contaminated sample Change buffers Only use buffers that are leads to unusually high supplied by QIAGEN or QIAGEN authorized consumption of substrate distributors mixture noted as a high dye Use the zoom in function to check if any peaks presequencing signal have been generated select a section of Pyrogram with the left mouse button Very high peaks PyroMark Q24 Validation Follow the instruction in the protocols for Oligo not correctly preparing the PyroMark Q24 Validation Oligo prepared Make sure to dilute the PyroMark Q24 Validation Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water
24. lity shown as a blue bar in the lower field of the well and with C indicated in a blue rectangle in the Pyrogram output C 5196 T 4996 250 200 150 100 0 E S C T G A C T G T G 5 Figure 5 Example of AQ assay result from a 50 mix tube CO 1 22 PyroMark Q24 Validation Oligo Handbook 09 2009 2 Determine the single peak heights Ideally the peaks should be between 30 10 RLU for samples with 0 5 picomoles of template and above 120 40 RLU for samples with 2 picomoles of template To obtain peak height values select Export Peak Heights from the Tools menu Save the data in a suitable format csv or tsv Open this file in Microsoft Excel Delimited and calculate the mean single peak height and background for each well as described below 3 Select the AQ CpG Analysis Results from the Reports menu to open the analysis result report 4 Save the data in a suitable format csv or tsv 5 Open the dota file in the Software 6 Prepare a table with expected and actual values An example is shown in Table 7 on page 24 7 Analyze the linearity according to the software instructions An example of linearity analysis is shown in Figure 6 on page 25 EP EEE WMM P PPPPPFPYENEDD SER PyroMark Q24 Validation Oligo Handbook 09 2009 23 Table 7 Expected and actual C values Tube label Sample Expected C Actual C A 5 6 22 2 5 6 17 3 5 5 06 E 1 16 3 18 20 2 16 3 17 90 3 16 3 18 12
25. n com 2009 QIAGEN all rights reserved www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 3865 3865 Fax 021 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 ltaly Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Or
26. ng primer is required The beads are released into PyroMark Annealing Buffer 11 Place PCR Plate 1 and a PyroMark Q24 Plate on the worktable of the PyroMark Q24 Vacuum Workstation see Figure 3 Ensure that the plate is in the same orientation as when samples were loaded Figure 3 Placement of PCR plate and PyroMark Q24 Plate on the PyroMark Q24 Vacuum Workstation The marked positions contain 7096 ethanol 1 PyroMark Denaturation Solution 2 PyroMark Wash Buffer 3 and high purity water 4 5 P Parking position 12 Turn on the pump and apply vacuum to the vacuum tool by opening the vacuum switch 13 Carefully lower the filter probes into the PCR plate to capture the beads containing immobilized template Hold the probes in place for 15 s Take care when picking up the tool Sepharose beads sediment quickly If more than 1 min has elapsed since the plate was agitated agitate again for 1 min before capturing the beads 14 Transfer the tool to the trough containing 7096 ethanol trough 1 Flush the filter probes for 5 s 15 Transfer the tool to the trough containing PyroMark Denaturation Solution trough 2 Flush the filter probes for 5 s 18 PyroMark Q24 Validation Oligo Handbook 09 2009 16 17 18 19 20 21 22 23 24 25 26 27 Transfer the tool to the trough containing PyroMark Wash Buffer trough 3 Flush the filter probes for 10 s Raise the to
27. oMark Q24 Enzyme Mixture Substrate Mixture and Nucleotides PyroMark Annealing For annealing sequencing primer to 979009 Buffer 250 ml single stranded PCR product and for Pyrosequencing reaction PyroMark Binding For binding of biotinylated PCR product 979006 Buffer 200 ml to Sepharose beads PyroMark Denaturation For denaturation of double stranded 979007 Solution 500 ml PCR product into single stranded template DNA PyroMark Wash Buffer For washing of single stranded DNA 979008 concentrate 200 ml PyroMark Q24 Plate 24 well sequencing reaction plate 979201 100 PyroMark Q24 Cartridges for dispensing reagents 979202 Cartridge 3 Related products PyroMark Q24 Sequence based detection platform for 9001514 Pyrosequencing of 24 samples in parallel PyroMark Q24 Application software 9019062 Software PyroMark Q24 Vacuum Workstation for preparing single Varies Workstation stranded DNA from 24 samples For use with PyroMark Q24 PyroMark Q96 MD and PyroMark Q96 ID t For use with PyroMark Q24 Vacuum Workstation and PyroMark Q96 Vacuum Workstation 9001518 220V 9001516 110V 9001519 100V ES e2ee eee lt lt X X ESSREERSSSE e8 EE ZXSMA SERR 34 PyroMark Q24 Validation Oligo Handbook 09 2009 Product Contents Cat no PyroMark Vacuum Prep Reusable filter probes for PyroMark 979010 Filter Probe 100 Vacuum Workstation Q96 and Q24 PyroMark Control For installation check of system 979203 Oligo
28. ocols for Oligo not correctly preparing the PyroMark Q24 Validation Oligo prepared Make sure to dilute the PyroMark Q24 Validation Oligo in the dilution buffer as described in the protocols Make sure that the 10x dilution buffer provided is first diluted to 1x using high purity water b Incorrect sequence to Check that the correct sequence was typed in analyze or dispensation the Assay Setup order c Buffers or reagents Follow the instructions supplied with the incorrectly diluted or reagents Include an empty well containing incorrectly stored only PyroMark Annealing Buffer in your run to check if background peaks are coming from the nucleotides d Dispensation error seen Clean or replace the PyroMark Q24 Cartridge for example as split If the problem remains contact QIAGEN peaks Technical Services for contact information see back cover or visit www qiagen com Blocked PyroMark Q24 Nucleotides are not dispensed correctly due to a Cartridge blocked needle in the PyroMark Q24 Cartridge Clean the PyroMark Q24 Cartridge and check that it is working properly f Damaged PyroMark Q24 Discard the PyroMark Q24 Cartridge according Cartridge to federal state and local environmental regulations for disposal of laboratory waste ALE 28 PyroMark Q24 Validation Oligo Handbook 09 2009 9 Annealing time too long Small or missing peaks a 2 Eu 9 PyroMark Q24 Validation Oligo Handbook 09 2009 Insufficien
29. ol up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 4 Figure 4 Illustration of the vacuum tool raised to beyond 90 vertical While the tool is held over the PyroMark Q24 Plate close the vacuum switch on the tool Off Release the beads in the plate containing 25 pl PyroMark Annealing Buffer by shaking the tool from side to side Allow the filter probes to rest on the bottom of the wells With the vacuum switch closed Off transfer the tool to the trough containing high purity water trough 4 and agitate the tool for 10 s Wash the filter probes by lowering the probes into high purity water trough 5 and applying a vacuum Flush the filter probes with 70 ml high purity water Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes see Figure 4 Close the vacuum switch on the tool Off and place the tool in the Parking P position Repeat steps 9 23 for the remaining PCR plates Plate 2 Plate 3 Plate 4 Turn off the vacuum pump At the end of a working day liquid waste and remaining solutions should be discarded and the PyroMark Q24 Vacuum Workstation should be checked for dust and spillage see Appendix B page 32 Heat the 4 PyroMark Q24 Plates with the samples at 80 C for 2 min using a heating block and the prewarmed PyroMark Q24 Plate Holder Note It is possible to process 4 PyroMark Q24 Plates sequentially
30. om the analysis should be analyzed by validated statistical software The mean and standard deviation for each mix is calculated based on the 8 replicates An example of data can be found in Table 8 Table 8 Results of bias and reproducibility determination Performance Expected C Actual C Standard deviation Bias 5 5 2 0 2 0 2 50 52 7 0 7 Dey 95 95 2 0 5 0 2 These values are given as an example only Actual values must be determined 8 Test for intermediate precision Intermediate precision can be tested using the same mixes in combination with the desired level of variation in terms of operator instrument and other reagents e RLL LUMtf MI TIITII TI TI PRE PEER PyroMark Q24 Validation Oligo Handbook 09 2009 27 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www qgiagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information or protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Refer to the PyroMark Q24 User Manual for general troubleshooting of the instrument Comments and suggestions Poor or incorrect sequence a PyroMark Q24 Validation Follow the instruction in the prot
31. roMark Q24 Cartridge with the label facing out Push the cartridge in fully and then push it down 46 Ensure the cartridge is properly inserted and close the gate 47 Open the plate holding frame and place the PyroMark Q24 Plate Plate 2 on the heating block 48 Close the plate holding frame and the instrument lid 49 Insert the USB memory stick containing the run file into the USB port at the front of the instrument Do not remove the USB stick before the run is finished 50 Select Run in the main menu using the and screen buttons and press OK 51 Select the run file Linearity 2picomol using the and screen buttons To view the contents of a folder select the folder and press Select To go back to the previous view press Back 52 When the run file is selected press Select to start the run 53 When the run is finished and the instrument confirms that the run file has been saved to the USB memory stick press Close 54 Repeat steps 38 53 for the remaining PyroMark Q24 Plates Plate 3 Plate 4 For Plate 3 use the Run file saved as BiasRepeatability O 5picomol For Plate 4 use the Run file saved as BiasRepeatability 2picomol 55 Remove the USB memory stick 56 Discard the PyroMark Q24 Plates and clean the PyroMark Q24 Cartridge see the PyroMark Gold Q24 Reagents Handbook PyroMark Q24 Validation Oligo Handbook 09 2009 21 Protocol 5 Analysis of Lineari
32. t amount of template for immobilization Not enough enzyme or substrate for all wells Wells marked in the Run Setup do not agree with sample placement in the plate One or more of the nucleotide compartments in the PyroMark Q24 Cartridge not correctly filled with reagents or nucleotides Dispensation error seen for example as split peaks Blocked PyroMark Q24 Cartridge Damaged PyroMark Q24 Cartridge Comments and suggestions Carry out annealing for the correct time and at the temperatures described in the protocols Make sure to dilute the PyroMark Q24 Validation Oligo correctly and use the amounts specified in the protocols Fill the PyroMark Q24 Cartridge according to the instructions in the Pre Run Information report Check that you loaded the PyroMark Q24 Plate correctly according to the Run Setup Make sure that sufficient reagents are added to the PyroMark Q24 Cartridge Follow the instructions for use supplied with the products Clean or replace the PyroMark Q24 Cartridge If the problem remains contact QIAGEN Technical Services for contact information see back cover or visit www qiagen com Nucleotides are not dispensed correctly due to a blocked needle in the PyroMark Q24 Cartridge Clean the PyroMark Q24 Cartridge and check that it is working properly Enzymes or substrates are not dispensed correctly due to a blocked PyroMark Q24 Cartridge as indicated by a missing presequencing signa
33. the samples in a random pattern in the PyroMark Q24 Plate An example of a random pattern is given in Table 1 and Table 2 where the letters refer to the mixes in Table 3 see Protocol 3 Preparation of PyroMark Q24 Validation Oligo Mixes and Dilution Series Enter C as Sample ID E Two Run Setups have to be prepared for each test one for 0 5 picomoles and one for 2 picomoles Procedure 1 Create 2 Run Setups for linearity determination by importing the Assay Setup assay parameters to the appropriate number of plates and wells as shown in Table 1 Save the Run Setups as Linearity 0 5picomol and Linearity 2picomol To add an assay to a well you can either m Right click the well and select Load Assay from the context menu m Select the assay in the shortcut browser and click and drag the assay to the well A well is color coded according to the assay type loaded onto the well For more information on how to create a Run Setup file see the PyroMark Q24 Software User Guide Table 1 Plate setup for linearity determination 3 C w gt O gt nm Q ON gt O m s Tola O O UO Q o PyroMark Q24 Validation Oligo Handbook 09 2009 11 2 Create 2 Run Setups for bias and repeatability determination by importing the Assay Setup parameters to the appropriate number of plates and wells as shown in Table 2 Save the Run Setups as BiasRepeatability 0 5picomol and BiasRepeatability 2picomol
34. ture The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish PyroMark Q24 Validation Oligo Handbook 09 2009 Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www giagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding PyroMark Q24 Validation Oligo or QIAGEN products in general please do not hes
35. ty The linearity of the assay can be tested at 2 levels E according to the Clinical Laboratory Standards Institute Guideline EP6 A as recommended by the Standard EN 13612 and using validated software or E by simple linear regression analysis Claimed performance for both CpG and AQ modes according to EP6 A For 0 5 2 picomoles of the PyroMark Q24 Validation Oligo using the method described here the method has been demonstrated to be linear from 5 to 95 C within an allowed nonlinearity of 3 percentage units in this interval Linearity according to CLSI EP6 A This method involves fitting linear and polynomial equations to the data The method then determines if the fit of the polynomial equation is significantly better than that of the linear equation in which case the data is nonlinear However acceptance limits may be set for nonlinearity to match the practical needs of the assay These are included in the analysis of the data to determine if any nonlinearity that is detected is acceptable There are a number of software products on the market to analyze data according to EP6 A The Software can be validated using for example datasets from the National Institute of Standards and Technology USA www nist gov Procedure 1 Open the run files for Linearity O 5picomol and Linearity 2picomol in the PyroMark Q24 Software and analyze all wells All wells expect the negative controls should be given Passed qua
36. uct signifies the agreement of any purchaser or user of the PyroMark Q24 Validation Oligo to the following terms 1 The PyroMark Q24 Validation Oligo may be used solely in accordance with the PyroMark Q24 Validation Oligo Handbook and for use with components contained in the Product only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this product with any components not included within this Product except as described in the PyroMark Q24 Validation Oligo Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Product and or their use s do not infringe the rights of third parties This product and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated QV expo uo CS The purchaser and user of the product agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Product and or its components For updated license terms see www giage
37. w qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor Cited references 1 SS ISO 5725 1 Accuracy trueness and precision of measurement methods and results Part 1 General principles and definitions 2 White H E Durston V J Harvey J F and Cross N C 2006 Clin Chem 52 1005 3 Tost J Dunker J and Gut I G 2003 Biotechniques 35 152 4 Colella S Shen L Baggerly K A Issa J P and Krahe R 2003 Biotechniques 35 146 5 Uhlmann K Brinckmann A Toliat M R Ritter H and N rnberg P 2002 Electrophoresis 23 4072 6 Neve B Frougel P Corset L Vaillant E Vatin V and Boutin P 2002 Biotechniques 32 1138 7 Wasson J Skolnick G love Gregory L and Permutt M A 2002 Biotechniques 32 1144 8 Gruber J D Colligan P B and Wolford J K 2002 Hum Genet 110 395 9 Clinical and Laboratory Standards Institute document EP6 A Evaluation of the linearity of quantitative measurement procedures a statistical approach approved guideline 10 EN 13612 Performance evaluation of in vitro diagnostic medical devices European Committee for Standardization PyroMark Q24 Validation Oligo Handbook 09 2009 33 Ordering Information Product Contents Cat no PyroMark Q24 For performance check of system 979204 Validation Oligo Accessories PyroMark Gold Q24 For 5 x 24 samples for use on the 971802 Reagents 5 x 24 Pyr
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