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TaqMan® Human Endogenous Control Plate

1. Note The columns of the selected cells contain the C values for the wells in row A of the TaqMan Human Endogenous Control Plate Select Edit gt Copy From the Window menu select the new spreadsheet The new spreadsheet file reappears Click on cell B2 Select Edit gt Paste Excel pastes the data into the new spreadsheet Using the cut and paste procedure from steps 1 6 copy the C values of the remaining wells into the new spreadsheet as shown below Select and copy the following cells 5700 results file 7700 results file Paste to cells D14 D25 F14 F25 C2 C13 D26 D37 F26 F37 D2 D13 D38 D49 F38 F49 E2 E13 D50 D61 F50 F61 F2 F13 D62 D73 F62 F73 G2 G13 D74 D85 F74 F85 H2 H13 D86 D97 F86 F97 12 113 Calculating Relative Quantification 5 7 To transfer data from the results file to the Cy table continued Step Action 8 Choose one of the following If the baseline and or threshold values were set Then once for all targets go to Deleting Invalid CT Values on page 5 10 separately for the targets as done in Setting Multiple Thresholds on page 4 7 or Appendix A Troubleshooting Early Amplification using the figure below as a reference replace the Cy values for the invalid wells as follows a Open the second results file
2. W gt O T m O DECOCO DO 6 6 6 ee COC ee eee JOO ODOOO QOOOOO OO CO Oe Ce Ce OOOO OOOO ac i fo CeCe Ce ee QOOOOO OO OOOO0O0O00O OOOO OOOO JJOO i i N Column Control Assay Abbreviation 1 Internal Positive Control IPC 2 18S rRNA 18S 3 Acidic ribosomal protein huPO 4 Beta actin hu A 5 Cyclophilin huCYC 6 Glyceraldehyde 3 phosphate dehydrogenase huGAPDH 7 Phosphoglycerokinase huPGK 8 Bo Microglobulin hu 2m 9 B Glucronidase huGUS 10 Hypoxanthine ribosyl transferase huHPRT 11 Transcription factor IID TATA binding protein huTBP 12 Transferrin receptor huTfR Note See Appendix B About These Assays for a list of the TaqMan assays and their functions Procedure The following diagram is a simplified overview of this protocol Flowchart Prepare total RNA samples and RT reaction mix RT thermal cycling Prepare PCR reaction mix and load the TaqMan Human Endogenous Control Plate PCR thermal cycling 4 Data analysis set baseline and threshold values Export analyzed data Spreadsheet analysis Graph the results and r 1 I i select a control Introduction 1 5 How TaqMan The TaqMan Human Endogenous Control Plate kit evaluates RNA Endogenous expression in a two step reverse transcription polymerase chain Control Assays reaction
3. 17 Load the plate into your thermal cycler and begin thermal cycling IMPORTANT Remove the 96 well reaction plate immediately after thermal cycling is complete The cDNA can be used immediately for PCR amplification or stored at 15 to 25 C for later use Reverse Transcription 2 7 Reverse Transcription for the 18S Amplicon Overview Recommended Template Template Quality Template Quantity 2 8 Reverse Transcription Synthesis of cDNA from total RNA samples is the first step in the two step RT PCR gene expression quantification experiment In this step random hexamers from the TaqMan Reverse Transcription Reagents P N N808 0234 prime total RNA samples for reverse transcription using MultiScribe Reverse Transcriptase Use only human total RNA samples to generate cDNA for the TaqMan Human Endogenous Control Plate The following table lists the known template incompatibilities Template Explanation Poly At The 18S rRNA endogenous control assay cannot accurately evaluate cDNA generated from poly A RNA samples because most of the rRNA has been removed from them Non human Except for 18S rRNA and the IPC all assays on the TaqMan Human Endogenous Control Plate are human specific The quality of your results is directly related to the purity of your RNA template Therefore use only well purified samples with the TaqMan Human Endogenous Control Plate Because ribonucleas
4. J M 1995 Genomic structure of the human TATA box binding protein TBP Gene 161 277 282 Dodge G R Kovalszky I Hassell J R and lozzo R V 1990 Transforming growth factor B alters the expboression of heparan sulfate proteoglycan in human colon carcinoma cells J Biol Chem 265 18023 18029 Forster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Annals of Physics Leipzig 2 55 75 Greenberg M E Greene L A and Ziff E B 1985 Nerve growth factor and epidermal growth factor induce rapid transient changes in proto oncogene transcription in PC12 cells J Biol Chem 260 14101 14110 Gussow D Rein R Ginjaar l et al 1987 The human B2 microglobulin gene Primary structure and definition of the transcriptional unit J Immunol 139 3132 3138 Holland P M Abramson R D Watson R and Gelfand D H 1991 Detection of specific polymerase chain reaction product by utilizing the 5 53 exonuclease activity of Thermus aquaticus DNA polymerase Proc Natl Acad Sci USA 88 7276 7280 C 2 References Innis M A Myambo K B Gelfand D H and Brow M A 1988 DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA Proc Nail Acad Sci USA 85 9436 9440 Koletsky A J Harding M W and Handschumacher R E 1986 Cyclophilin distribution and variant properties in normal and neoplastic tissues J Immunol 137 1054 10
5. Protein Sequencing corelab appliedbiosystems com Peptide and DNA Synthesis Biochromatography tsupport appliedbiosystems com PerSeptive DNA PNA and Peptide Synthesis systems FMAT 8100 HTS System CytoFluor 4000 Fluorescence Plate Reader Voyager Mass Spectrometers Mariner Mass Spectrometers LC MS Applied Biosystems MDS support sciex com Sciex Chemiluminescence Tropix tropix appliedbiosystems com Technical Support D 1 Hours for Technical Support D 2 Technical Support In the United States and Canada technical support is available at the Telephone following times Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time To Contact Technical Support by Telephone or Fax In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Dial Fax Dial ABI PRISM 3700 DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 2 then 1 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 2 then 2 1 650 638 5981
6. 2 319 9788 Europe Austria Wien 43 0 1 867 35 75 0 43 0 1 867 35 75 11 Belgium 32 0 2 532 4484 32 0 2 582 1886 Czech Republic and 420 2 35365189 420 2 35364314 Slovakia Praha Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Poland Lithuania Latvia 48 22 866 40 10 48 22 866 40 20 and Estonia Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 502 935 8888 7 502 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 838 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 Technical Support D 5 To Reach Technical Support Through the Internet D 6 Technical Support Region Telephone Dial Fax Dial The Netherlands Nieuwerkerk a d IJssel 31 0 180 392400 31 0 180 392409 or 31 0 180 392499 United Kingdom 44 0 1925 825650 44 0 1925 282502 Warrington Cheshire All other cou
7. 4 11 Calculating Relative Quantification Overview About This This chapter explains how to calculate relative quantification values Chapter from C values with the use of a spreadsheet application such as Microsoft Excel Applied Biosystems recommends using a professional spreadsheet software package to analyze the results from the TaqMan Human Endogenous Control Plate Although calculation of relative quantification values can be done manually spreadsheet packages speed the process considerably In This Chapter The following topics are discussed in this chapter Topic See Page Exporting and Viewing the Results File 5 2 Calculating the Relative Quantification Using a Spreadsheet 5 5 Interpreting Results 5 17 Calculating Relative Quantification 5 1 Exporting and Viewing the Results File Creating a To analyze data from the TaqMan Human Endogenous Control Plate Results File export the results to a results file The SDS software can export raw data from a sequence detection run in formats that are compatible with most spreadsheet applications The type of file the software exports depends on the model instrument used to collect the data Instrument Exported Format ABI PRISM 7700 Instrument Tab delimited text file GeneAmp 5700 Instrument Comma separated text file csv Exporting Results from a GeneAmp 5700 Sequence Detection System To export the data from the endogenous control gene e
8. RT PCR The figure below illustrates the assay steps Work Extension of primer on mRNA E 3 mRNA RT _ f CDNA Random Step Hexamer Synthesis of 1st cDNA strand a 5 CDNA Extension of primer on cDNA 3 5 Forward Primer Synthesis of 2nd cDNA strand 3 Cycle 1 PCR Step Ls 5 m 3 PCR amplification of cDNA Forward Primer 5 3 5 Cycle 2 Reverse Primer GR1312 In the RT step cDNA is reverse transcribed from total RNA samples using random hexamers from the TaqMan Reverse Transcription Reagents In the PCR step products are synthesized from cDNA samples using the TaqMan Universal PCR Master Mix 1 6 Introduction Basics of the The PCR reaction exploits the 5 nuclease activity of AmpliTaq Gold 5 Nuclease Assay DNA Polymerase to cleave a TaqMan probe during PCR The TaqMan probe incorporates a VIC reporter dye at the 5 end of the probe and a quencher dye at the 3 end of the probe During the reaction cleavage of the probe separates the VIC reporter dye and the quencher dye which results in increased fluorescence of the reporter Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye as shown in the figure below Polymerization R Reporter Q Quencher Forward R Q Primer Probe 5 3 3 5 5 3 ae rimer Strand displacement 5 3 3 5 5 g 5 Cleavage
9. and the calibrator sample 2 10 Reverse Transcription To prepare the reverse transcription reactions continued Step Action 8 Pipette 65 25 uL of the reaction mix from step 1 to each MicroAmp Reaction Tube from step 7 10X RT buffer ie MgClo __ dNTPs mixture Random hexamers e MultiScribe reverse Transcriptase _ RNase inhibitor 65 25 uL 65 25 uL 65 25 uL 65 25 uL Calibrator Sample 1 Sample 2 Sample 3 9 Transfer 34 75 uL of each dilute total RNA sample to the corresponding MicroAmp Reaction Tube 10 Cap the reaction tubes and gently tap each to mix the reactions 11 Briefly centrifuge the tubes to force the solution to the bottom and to eliminate air bubbles from the mixture 12 Transfer each reaction to MicroAmp Optical tubes or wells of a MicroAmp Optical 96 Well Reaction plate 13 Cap the MicroAmp Optical tubes or plate 14 Centrifuge the plate or tubes to spin down the contents and eliminate air bubbles from the solutiions Reverse Transcription 2 11 Thermal Cycling To conduct reverse transcription thermal cycling Step Action 1 Load the reactions into a thermal cycler 2 Program your thermal cycler with the following conditions Reverse St Hexamer Reverse Transcriptase ep Incubationa Transcription Inactivation HOLD HOLD HOLD Temp 25 C 37 C 95 C Time 10 min 60 min 5 min Volume 100 uL a When using
10. lists the storage conditions for the TaqMan Human Guidelines Endogenous Control Plate Kit and reagents used in this protocol Kit Component Reagent Storage Conditions TaqMan Human Endogenous Control Plate 2 to 8 C dark TaqMan Universal PCR Master Mix 2 to 8 C dark TaqMan Human Control Total RNA 15 to 25 C IMPORTANT Do not remove the TaqMan Human Endogenous Control Plate from its packaging until ready to use Excessive exposure to light damages the fluorescent probes 1 10 Introduction Materials and Equipment Not Included Some equipment and materials are required in addition to the reagents supplied with the TaqMan Human Endogenous Control Plate Many of the items listed are available from major laboratory suppliers MLS Equivalent sources are acceptable where noted Sequence Detection Systems Source ABI PRism 7700 Sequence Detection System GeneAmp 5700 Contact your local Applied Biosystems sales office for the instrument best suited to meet Sequence Detection System your needs User supplied materials Materials Source MicroAmp Optical 96 Well Reaction Plate Optical Caps Applied Biosystems P N 403012 Note The MicroAmp Optical 96 Well Reaction Plate may be sealed with MicroAmp Optical Caps or ABI PRISM Optical Adhesive Cover The Optical Adhesive Cover must be used with a compression pad and applicator which are included
11. of cycles over which the software can calculate the baseline As the available baseline is compressed the amplification plots of the less abundant targets may appear to disperse This can lead to poor reproducibility and inaccurate quantification For example in the figure below the baseline is set correctly for the 18S amplification baseline is set for cycles 2 7 however the plots of the less abundant targets have become dispersed As a result Cy values from this plot are valid only for the 18S amplifications Plots of the 18S rRNA target Plots of less abundant targets dispersed Baseline cycles 2 7 The baseline in the figure below is reset for the less abundant targets baseline is set for cycles 3 15 Notice that the amplification plots of these targets are now well pronounced and allow the SDS software to determine accurate C values In contrast the plots of 18S targets now exhibit a sigmoidal curve and do not yield valid data points Troubleshooting Early Amplification A 1 Plots of less abundant targets Plots of the 18S rRNA target Baseline cycles 3 15 How to Correct When early amplification of the 18S rRNA target interferes with the for Early detection of less abundant genes set the baseline and threshold values Amplification independently for each group of plots The following procedure explains how to configure each group of plots independently and export the data The results from
12. plays an important role in the metabolic salvage of purines in mammalian cells Transcription Factor IID TATA Binding Protein The TATA binding protein is constitutively expressed in many tissues and cells at low levels It is required for transcription directed by RNA polymerases Il and III Chalut et al 1995 huTBP Transferrin Transferrin receptor mediates cellular iron uptake and is expressed at low levels Receptor in both tissues and cells The expression of the receptor on the cell surface huTfR correlates with cellular proliferation being highest on rapidly dividing cells and much lower on resting cells and more terminally differentiated cell types McClelland et a 1984 As shown in the figure in Demonstrating Performance with TaqMan Human Control Total RNA on page 5 20 transferrin receptor exhibits the lowest level of gene expression when evaluating TaqMan Human Control Total RNA using the TaqMan Human Endogenous Control Plate B 2 About These Assays References Allen R W Trach K A and Hoch J A 1987 Identification of the 37 kDa protein displaying a variable interaction with the erythroid cell membrane as glyceraldehyde 3 phosphate dehydrogenase J Biol Chem 262 649 653 Bonini J A and Hofmann C 1991 A rapid accurate nonradioactive method for quantitating RNA on agarose gels Biotechniques 11 708 710 Chalut C Gallois Y Poterszman A Moncollin V and Egly
13. random hexamers for first strand cDNA synthesis a primer incubation step 25 C for 10 min is necessary to maximize primer RNA template binding 3 Begin reverse transcription IMPORTANT After thermal cycling store all cDNA samples at 15 to 25 C 2 12 Reverse Transcription PCR Overview About This This chapter covers PCR or the amplification of cDNA PCR is the Chapter second step in the two step RT PCR experiment as described in How TaqMan Endogenous Control Assays Work on page 1 6 In this step AmpliTaq Gold DNA polymerase amplifies cDNA synthesized from the original total RNA sample Note See Basics of the 5 Nuclease Assay on page 1 7 for more information on AmpliTaq Gold DNA polymerase and the 5 nuclease assay In This Chapter The following topics are discussed in this chapter Topic See Page Preparing the Sequence Detection System for PCR 3 2 Preparing and Running the PCR Reactions 3 5 PCR 3 1 Preparing the Sequence Detection System for PCR Instruments Configuring the ABI PRISM 7700 Software for the VIC Dye Programming the Sequence Detector for PCR IMPORTANT Because the data acquired during the PCR is needed for analysis you must use one of the following sequence detectors for PCR ABI PRISM 7700 Sequence Detection System GeneAmp 5700 Sequence Detection System If your ABI PRISM 7700 Sequence Detection System is not calibrated for the VIC
14. the results files are combined during spreadsheet analysis To set the baseline and threshold separately Step Action 1 From the amplification plot deselect the 18S wells that amplify during the very early cycles of the PCR 2 Following the guidelines in Guidelines for Setting the Baseline on page 4 2 set the baseline for those plots that amplify during the later cycles of the PCR 3 Following the guidelines in Guidelines for Setting the Threshold on page 4 7 set the threshold for the plots that amplify during the later cycles of the PCR 4 Export the data as explained in Exporting and Viewing the Results File on page 5 2 The software saves the data The results in the file are valid only for the wells that amplify during the later cycles 5 From the amplification plot deselect the wells that amplify during the later cycles of the PCR 6 Following the guidelines in Guidelines for Setting the Baseline on page 4 2 reset the baseline for the 18S wells that amplify during the very early cycles of the PCR 7 Following the guidelines in Guidelines for Setting the Threshold on page 4 7 set threshold for the 18S wells that amplify during the very early cycles of the PCR A 2 Troubleshooting Early Amplification To set the baseline and threshold separately continued Step Action 8 Export the data as explained in Exporting and Viewing the Re
15. to demonstrate the Performance with performance of the TaqMan Human Endogenous Control Plate The TaqMan Human figure below shows a typical gene expression profile for the sample Control Total RNA huPGK huB2m huGUS huHPRT huTBP huTfR I Q a lt 5 5 20 Calculating Relative Quantification To generate the profile shown above Step Action 1 Perform the reverse transcription step as described in Reverse Transcription for All Amplicons Except 18S on page 2 4 using the TaqMan Human Control Total RNA 10 ng per well 2 Perform the PCR step as described in Chapter 3 PCR configuring the plate with duplicate wells for the control sample 3 Analyze and export the data See Chapter 4 Data Analysis 4 Construct a AC table and import data to it by following the procedures on pages 5 4 to 5 7 5 Select the column of cells containing the Cy data for the Human Control Total RNA 6 Select Insert gt Chart gt On This Sheet 7 Click the selected data Follow the instructions as directed by the wizard Calculating Relative Quantification 5 21 Troubleshooting Early Amplification Effects of Early In rare cases the amplification of the 18S assay can interfere with the Amplification of detection of less abundant targets When amplification of the 18S target the 18S Assay reaches a detectable level at a very early cycle it limits the number
16. 3 gz 2 On gas nS e a a a Vg i gt 4 00 wo eS 5 00 D lt ACt Sample 1 E ACt Sample 2 ACt Sample 3 The Relationship One AC is equal to a twofold difference in initial template Between AC and concentration This relationship is shown with the following equation Gene Expression X Xp 1 Ey Where Xn Copy number at cycle n Ey Amplicon efficiency Xo Copy number at cycle 0 n Cycle number Because amplicons designed and optimized according to Applied Biosystems guidelines have equivalent efficiencies approaching 100 it can be stated that Ey 1 Also because we are interested in the difference in initial template for one cycle it can be stated that n 1 5 18 Calculating Relative Quantification Choosing an Endogenous Control Substituting values for the appropriate variables the equation becomes X Ks 1 2X Choose the control with the least variation in AC levels Ideally the best control is expressed at a constant level in all samples regardless of cell cycle cell type or tissue Because the AC indicates the level of gene expression relative to the calibrator the AC values of a good control do not vary much from zero It is important to remember that a difference of one cycle equates to a twofold difference in initial template For example a control with AC values that vary over a two cycle range would have nearly a fourfold difference in expression l
17. 59 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lakowicz J R 1983 Principles of Fluorescence Spectroscopy ed New York Plenum Press xiv 496 pp Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 McClelland A K hn L C and Ruddle F H 1984 The human transferrin receptor gene genomic organization and the complete primary structure of the receptor deduced from a cDNA sequence Cell 39 267 274 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction Methods Enzymol 155 335 350 Mutimer H Deacon N Crowe S and Sonza S 1998 Pitfalls of processed pseudogenes in RT PCR Biotechniques 24 585 588 Okubo K and Matsubara K 1997 Complementary DNA sequence EST collections and the expression information of the human genome FEBS Lett 403 225 229 Oshima A Kyle J W Miller R D et al 1987 Cloning sequencing and expression of cDNA for human B glucuronidase Proc Natl Acad Sci USA 84 685 689 Patel P I Framson P E Caskey C T and Chinault A C 1986 Fine structure of the human hypoxanthine phosphoribosyltransferase gene Mol Cell Biol 6 393 403 Raff T van der Giet M Endemann D Wiederholt T and Paul M 1997 Design and testing of B actin primers for RT
18. 700 7700 Sequence Detector 5700 Quantitation Real Time 3 2 PCR To configure the PCR plate document continued Step Action 3 Choose from one of the following If using an Then ABI PRISM 7700 Sequence From the Dye Layer menu select VIC Detection System Note If VIC does not appear on the Dye Layer menu the instrument is not calibrated for the VIC dye See Configuring the ABI Prism 7700 Software for the VIC Dye on page 3 2 for more information GeneAmp 5700 Sequence a From the Primer Probe Setup dialog box create the Detection System following primer probe entry Acronym TAQ1 Description TaqMan VIC b Apply the probe to all wells 4 Configure the plate document as shown in the figure below i UNKN huHPRT huHPRT huHPRT Note See Appendix B About These Assays for a list of target names abbreviations and descriptions PCR 3 3 To configure the PCR plate document continued Step Action 5 Program the thermal cycler with the following conditions AmpliTaq Gold UNG Activation Activation PCR HOLD HOLD CYCLE 40 cycles Denature Anneal Step Extend Temp 50 0 C 95 0 C 95 0 C 60 0 C Time 2 min 10 min 15 sec 1 min Volume 50 uL a Required for optimal AmpErase UNG activation b Required for optimal AmpliTaq Gold DNA Polym
19. A ES ER ry verte i all Caleb g bor E a E TE 7E Re i ra 03 07 0 N Select Insert gt Chart gt On This Sheet The Excel chart wizard requests data for the new graph 3 Click the selected data The chart wizard prompts you for information 4 Follow the instructions as directed by the wizard Calculating Relative Quantification 5 17 Interpreting the The results of the Endogenous Control Plate are expressed in AC Gene Expression greater than or less than the calibrator ACz Thus the calibrator serves Profile as a baseline for the assays and is shown as zero on the graph Samples with positive AC values have initial template concentrations higher than that of the calibrator sample Samples with negative AC values have initial template concentrations lower than that of the calibrator sample Note See About the AC Equation on page 5 13 for more information The plot below illustrates a typical gene expression profile 2 00 IPC 18S huPO huBA huCYC huGAPDH huPGK huB2m huGUS huHPRT huTBP huTfR N Lo 2 R 1 00 2 5 S zS s 8 8 coo Q 0 00 5 o Boe aof 3S B85 m T sD 8 e oO P e 2 lo lo a 3 S gt 2 00 ird 2 a 2 3 2 z
20. CR Step Action 1 Pipette 650 uL of TaqMan Universal PCR Master Mix 2X into each of 4 microcentrifuge tubes three test samples and a calibrator sample TaqMan PCR ___ Universal Master Mix 2X 650 uL 650 uL 650 uL 650 uL Calibrator Sample 1 Sample 2 Sample 3 2 Dilute three test samples and a calibrator sample from the RT step to a volume of 650 uL with RNase free water Volume uL Component Well Tube cDNA from RT step X y RNase free water 25 x 650 y Total volume 25 650 a Volume includes reaction mix for one test sample or the calibrator sample enough to fill 24 wells of the endogenous control plate b Includes volume for 24 wells IMPORTANT Slowly and carefully remove the caps from the reaction plate or tubes to avoid contamination of the reverse transcription products To perform the PCR continued Step Action 3 Pipette 650 uL of each cDNA sample to a microcentrifuge tube containing TaqMan Universal PCR Master Mix anret Sampie 1 RNase free RNase free 650 uL 650 uL 650 uL 650 uL S 650 uL 650 uL 650 uL 650 uL Universal Universal Universal Universal Master Mix Master Mix Master Mix Master Mix Calibrator Sample 1 Sample 2 Sample 3 Sampe 2 Sampe 3 RNase free RNase free H20 7 Cap the microcentrifuge tubes and mix the solutions by gentle inversion Centrifuge the tubes to spin down the
21. D1 Avg Ct Calibrator G1 Avg Ct Sample 1 J1 Avg Ct Sample 2 M1 Avg Ct Sample 3 Calculating Relative Quantification 5 11 To add Average C columns to your C table continued Step Action 3 Average the C values of duplicate calibrator and sample wells by typing the following formulas into the specified cells Click cell Type D2 AVERAGE B2 C2 Excel averages the C values of cells B2 and C2 and displays it in cell D2 G2 AVERAGE E2 F2 Excel averages the C values of cells E2 and F2 and displays it in cell G2 J2 AVERAGE H2 12 Excel averages the C values of cells H2 and 12 and displays it in cell J2 M2 AVERAGE K2 L2 Excel averages the C values of cells K2 and L2 and displays it in cell M2 4 Copy the formulas entered into the spreadsheet in the previous step and paste them to the remaining cells of each column as follows Select and copy cell Paste to cells D2 D3 D13 G2 G3 G13 J2 J3 J13 M2 M3 M13 Excel averages the C values for the two cells to the left of each copied cell and displays the averaged Cr Average C values AverageC values Average C values Average Cy values of duplicate of duplicate wells of duplicate wells of duplicate wells calibrator wells for Sample 1 for Sample 2 for Sample 3 5 12 Calculating Relative Quantification About the AC Equation Constructing a AC Ta
22. Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 2 then 3 1 650 638 5981 Integrated Thermal Cyclers ABI PRISM 877 and Catalyst 800 instruments 1 800 831 6844 then press 2 then 4 1 650 638 5981 ABI PRism 3100 Genetic Analyzer 1 800 831 6844 then press 2 then 6 1 650 638 5981 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 3 then 1 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 3 then 2 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 7900 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Voyager MALDI TOF Biospectrometry Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 1 then 3 1 508 383 7855 Technical Support D 3 D 4 Technical Support Product or Product Area Telephone Dial Fax Dial Biochromatography BioCAD Workstations and POROS Perfusion Chromatography Products 1 800 899 5858 then press 1 then 4 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 1 then 5 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 1 then 5 1 508 383 7855 PNA Custom and Sy
23. MultiScribe Reverse Transcriptase RNase Inhibitor in a freezer until immediately prior to use Because the data acquired during the RT reaction is not needed for analysis any of the thermal cyclers listed below can be used ABI PRISM 7700 Sequence Detection System GeneAmp 5700 Sequence Detection System GeneAmp PCR System 9700 Thermal Cycler GeneAmp PCR System 9600 Thermal Cycler Performing the CHEMICAL HAZARD TaqMan Reverse Transcription Reagents may cause eye and skin irritation Always use adequate ventilation such as that provided by a fume hood Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and RT Reaction gloves To perform the RT reaction Step Action 1 In a 1 5 mL microcentrifuge tube prepare a reaction mix for all total RNA samples to be reverse transcribed Volume pL Per Reaction Component Sample Mix x4 Final Conc RNase free water See See E belowa belowa 10X RT Buffer 10 0 40 0 1X 25 mM MgCl 22 0 88 0 5 5 mM deoxyNTPs Mixture 20 0 80 0 500 uM per ANTP Random Hexamers 5 0 20 0 2 5 uM RNase Inhibitor 2 0 8 0 0 4 U uL MultiScribe Reverse 2 5 10 0 1 25 U uL Transcriptase 50 U L Totalb c 61 5 246 0 a The volume of RNase free water uL is 38 5 uL RNA sample volume in a 100 uL reaction b If changing the reaction volume make sure that the final proportions are consisten
24. O B20 huBA B21 huCYC B22 huGAPDH B23 huPGK B24 huB2m B25 huGUS B26 huHPRT B27 huTBP B28 huTfR 3 Type the following AC labels into the specified cells in the table Click on cell Type C16 ACt Sample 1 D16 ACt Sample 2 E16 ACt Sample 3 F16 Average ACt G16 ACt Calibrator The AC table appears as shown below a a ea a iH 1a En i EI iae r 3 han 26 10 hue Fa HENE 5 14 Calculating Relative Quantification Calculating AC To calculate AC values for the calibrator and samples Values Step Action 1 Type the following formulas into the specified cells Click cell Type C17 D2 G2 Excel subtracts the averaged C value for Sample 1 cell G2 from the averaged C value for the calibrator cell D2 D17 D2 J2 Excel subtracts the averaged C value for Sample 2 cell J2 from the averaged C value for the calibrator cell D2 E17 D2 M2 Excel subtracts the averaged C value for Sample 3 cell M2 from the averaged C value for the calibrator cell D2 F17 AVERAGE C17 D17 E17 Excel averages AC values for the three samples yielding an overall mean for the IPC endogenous control G17 D2 D2 Excel subtracts the averaged calibrator Cy cell D2 from itself to verify the calibrator The AC table appears as shown below Tar eH ME ede eee AI Hga i ara eee dL a2 G2 afiz 12 aba A eM ERECT abo o 7 hu Pit a buble T he GS 10 huHPe
25. PCR that do not co amplify processed pseudogenes Biotechniques 23 456 460 Rich B E and Steitz J A 1987 Human acidic ribosomal phosphoproteins PO P1 and P2 analysis of cDNA clones in vitro synthesis and assembly Mol Cell Biol 7 4065 4074 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Spanakis E 1993 Problems related to the interpretation of autoradiographic data on gene expression using common constitutive transcripts as controls Nucleic Acids Res 21 3809 3819 References C 3 Technical Support Technical Support Contacting Technical Support To Contact Technical Support by E Mail You can contact Applied Biosystems for technical support by telephone or fax by e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below Contact technical support by e mail for help in the following product areas Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems and pcrlab appliedbiosystems com PCR
26. TaqMan Human Endogenous Control Plate Protocol For Research Use Only Not for use in diagnostic procedures KS Bee ns Copyright 2001 2010 Applied Biosystems For Research Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER LIMITED LICENSE ABI PRISM and its design Applied Biosystems GeneScan and MicroAmp are registered trademarks of Life Technologies Corporation or its subsidiaries in the U S and certain other countries MultiScribe and VIC are trademarks of Life Technologies Corporation or its subsidiaries in the U S and certain other countries AmpErase AmpliTaq Gold GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems Inc AppleScript and Macintosh are registered trademarks of Apple Inc Microsoft is a registered trademark of Microsoft Corporation in the United States and or other countries All other trademarks are the sole property of their respective owners Contents Introduction OVCIVIEW fis pict cue Mak Gye ee yh epee ae SG ee Meee enh easy eect Gots 1 1 Control Plate Assay System 0 eee ee eee ee 1 2 Preventing Contamination 0 0 e eee eee cee 1 9 Materials and Equipment 0 0 0 eee ee eee eee eee 1 10 Salety sched studied ecules grade A Seade a E R aac A beaks yg toe eae 1 13 Reverse Transcription OVETVICW ise i Bae BE RAS ATR AREER be ESI ee BE 2 1 Sample Preparation 0 0 00 cece cee eee eee 2 2 Reverse Transcripti
27. a 3 3 5 5 3 lt I S Polymerization completed 3 5 m gt 3 5 5 3 ee 5 When the probe is intact the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer F rster 1948 Lakowicz 1983 During PCR if the target of interest is present the probe specifically anneals between the forward and reverse primer sites The 5 3 nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the Introduction 1 7 About AmpliTaq Gold DNA Polymerase TaqMan Universal PCR Master Mix 1 8 Introduction probe hybridizes to the target The probe fragments are then displaced from the target and polymerization of the strand continues The 3 end of the probe is blocked to prevent extension of the probe during PCR This process occurs in every cycle and does not interfere with the exponential accumulation of product The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR Because of these requirements any nonspecific amplification is not detected AmpliTaq Gold is a thermal stable DNA polymerase The enzyme has a 5 gt 3 nuclease activity but lacks a 3 5 exonuclease activity Innis et al 1988 Holland et al 1991 When using AmpliTagq Gold enzyme you can introduce Hot Star
28. alkali sensitive apyrimidic site in the DNA The enzyme has no activity on RNA or dT containing DNA Longo et al 1990 Please follow these recommended procedures Wear a clean lab coat not previously worn while handling amplified PCR products or during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect that they are contaminated Maintain separate areas dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Use positive displacement pipettes or aerosol resistant pipette tips Regularly clean benches and equipment with 10 bleach solution Introduction 1 9 Materials and Equipment Kit Components TaqMan Human Endogenous Control Plates are available in the following configurations P N Contents 4309920 Component P N TaqMan Human Endogenous Control Plates 2 TaqMan Universal PCR Master Mix 4304437 TaqMan Human Control Total RNA 4307281 TaqMan Human Endogenous Control Plate 4308134 Protocol 4309921 Component P N TaqMan Human Endogenous Control Plates 2 TaqMan Universal PCR Master Mix 4304437 Materials Storage The table below
29. and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Reverse Transcription 2 9 To prepare the reverse transcription reactions Step Action 1 In a1 5 mL microcentrifuge tube prepare a reaction mix for all total RNA samples to be reverse transcribed Volume uL Per Reaction Final Component Sample Mix x4 Conc RNase free water See See belowa belowa 10X RT Buffer 10 0 40 0 1X 25 mM MgCl 22 0 88 0 5 5 mM deoxyNTPs Mixture 20 0 80 0 500 uM per dNTP Random Hexamers 5 0 20 0 2 5 uM RNase Inhibitor 2 0 8 0 0 4 U uL MultiScribe Reverse 6 25 25 0 3 125 U uL Transcriptase 50 U uL Total 65 25 261 0 a The volume of RNase free water uL is 34 75 RNA sample volume in a 100 uL reaction b If changing the reaction volume make sure the final proportions are consistent with the recommended values above 2 Label four 1 5 mL microcentrifuge tubes for the three test samples and a calibrator sample 3 Transfer 60 ng 2 pg up to 34 75 uL of each total RNA sample to the corresponding microcentrifuge tube 4 If necessary dilute each total RNA sample to a volume of 34 75 uL with RNase free deionized water 5 Cap the tubes and gently tap each to mix the diluted samples 6 Briefly centrifuge the tubes to eliminate air bubbles in the mixture 7 Label four 0 2 mL MicroAmp Reaction Tubes for the three total RNA test samples
30. b Copy and paste the C values of the valid wells from the second file to the C table replacing the invalid values from the first results file c Deleting Invalid CT Values on page 5 10 5 8 Calculating Relative Quantification To transfer data from the results file to the Cy table continued Step Action The following figures illustrate the placement of the well data in the C table As shown the cells in the C table correspond to the 96 wells of the TaqMan Human Endogenous Control Plate ee ee ee 2 es ee eee ee ee 0 aee eak a a Ci d O C a n n E ata awn song Buia SUNS omuet tuiga E tal AM E ont En Ba eae eas m E om E Well 17 D Wan 2 E Wall an E AET EE EE WaT CE well 1A CE Wali Sd Ce wall al C aga ere neta A aE e ana po i an 7 WaT ZI E Wall E ei LE E TE i Ch ani 1 Ch wall Z2 CE Wah 54 Ch Wallan CA wall 58 Ea i well if wen si Ct well Sh C aia o gt well Se 15 wei Ganba Ch wal Sg th eiae f Well op EEI 1 2 3 4 5 6 7 8 9 10 11 12 O OQOQOQDOQOQOD cae 00000000 0 O eH a Well numbers 90000000A ame correspond to O OHOOQHOOO aris E 49 60 G1 ED Gs Gs CO E E Sample 2 TaqMan Human O0O000000000 ae 9OOOOOO games OOOO O OMOOeOwe e Calculating Relative Quantification 5 9 Deleting Invalid Cr Values Before averaging the C values of duplicate wells clear the data for all wells containing values outsi
31. ble Derivation of AC values from the average C values of the calibrator and samples is the final step in comparative gene expression analysis The following equation describes the AC calculation ACT sample AverageCy calibrator AverageCrsample The equation above uses the average C of the calibrator as a baseline for evaluating target gene expression in each sample Samples with initial template concentrations higher than the calibrator have lower average C values and yield positive numbers Samples with lower initial template concentrations have higher average C values and yield negative numbers To construct a AC table Step Action 1 Copy cells A1 A13 and paste into cells A16 A28 ea Ee Sa BS ed es ee es LL irae I Z1e3 zJ ZL 21 15 Z1 z 11 78 ho lai cl te cl ie giid 1509 2 4 13 0 We 15 te ME Ub m mat 2T 11 1 TI B40 Fa i mar IEJ IE 1 fa oa Paes oa oa 2475 a 3 1143 1743 15 EN i a m4 mI mn 2601 Ze PEt a7 5 ILIZ IT FEEF oa a ara oo he a Co fet SEE w fee He and Pa a PIM beet yy Eea Shee Sheers Steers a a fo 4 La Calculating Relative Quantification 5 13 To construct a AC table continued Step Action 2 Type the following labels into the specified cells in the table Click on cell Type B16 Target B17 IPC B18 18S B19 huP
32. contents and eliminate air bubbles from the solutions PCR 3 7 3 8 PCR To perform the PCR continued Step Action 6 Using a positive displacement pipette transfer the three test samples and a calibrator sample 50 uL aliquots to the wells of the TaqMan Human Endogenous Control Plate cDNA Master Mix RNase free H2O mixture 1 3 mL o N Calibrator gt wo O O OORM CO Ge OORM OCC ee OORM OC Re OORM CORE 010 00 OOS OO Ge OOS CO Ge OOM CORE OOM CORR OORM OOS OORS OO oje 00 I GR1280b Sample 3 Sample 2 Sample 1 cr Be e a Transfer 50 uL to each well 7 Seal the wells with MicroAmp Optical Caps or a MicroAmp Optical Adhesive Cover 8 Centrifuge the 96 well plate to spin down the contents and eliminate any air bubbles from the solutions 9 Load the TaqMan Human Endogenous Control Plate into your sequence detection system and begin thermal cycling Refer to the thermal cycling conditions on page 3 4 Data Analysis Overview About This This chapter covers data analysis which requires adjustment of the Chapter baseline and threshold values within the Sequence Detection Systems SDS software After the adjustments the data can be exported from the SDS software for spreadsheet analysis IMPORTANT If the threshold value is set before the baseline the resulting C values may be invalid a
33. d safety guide is a separate document sent to all and Safety Guide customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Introduction 1 15 Reverse Transcription Overview About This This chapter covers reverse transcription RT a process in which Chapter CDNA is synthesized from total RNA samples Reverse transcription is the first step in the two step RT PCR gene expression quantification experiment as described in How TaqMan Endogenous Control Assays Work on page 1 6 In this step random hexamers from the TagqMan Reverse Transcription Reagents prime total RNA samples for reverse transcription using MultiScribe Reverse Transcriptase In This Chapter The following topics are discussed in this chapter Topic See Page Sample Preparation 2 2 Reverse Transcription for All Amplicons Except 18S 2 4 Reverse Transcription for the 18S Amplicon 2 8 Reverse Transcription 2 1 Sample Preparation Recommended Template RNA Template Preparation and Quality Recommended Quantity 2 2 Reverse Transcription Based on the template conflicts explained below Applied Biosystems recommends evaluating only human total RNA samples using the TaqMan Human Endogenous Control Plate The following table lists the known template incompatibilities Template Expla
34. de the given dynamic range of the assays Invalid C values may be the result of experimental error degraded samples pipetting inaccuracy If averaged using the C values from the duplicate wells the invalid data can skew the results and indicate an incorrect level of gene expression Guidelines for Deleting Invalid Cy Values Use the following guidelines to identify invalid data for deletion Guideline Description Use the 18S rRNA assay as an indicator of sample concentration and quality Because cells generally express 18S rRNA at extremely high levels the target is usually a good indicator of sample concentration Typically the 18S assay yields C values lt 22 If sample produces C values above 22 for the 18S assay it may not contain enough cDNA for accurate analysis Therefore the sample must be cleared from the spreadsheet as shown below The calibrator data must be deleted because the C values for the 18S assay cells B2 and C2 are greater than 22 cycles Look for individual outlying Cys Occasionally a single well produces a C outside the average for its target group Abnormalities of this kind are typically due to experimental error rather than differences in gene expression To obtain accurate C values for the sample the C must be cleared from the spreadsheet as shown below Caer ec Ces Coo Le Cl Lal ETE This value is I z163 eaS ro ZII z 11 7 1350 H z beyond the ave
35. dye it must be calibrated using the Sequence Detection Systems Spectral Calibration Kit P N 4305822 The kit provides the standards needed to configure the ABI PRISM 7700 Sequence Detector for use with products containing TaqMan VIC or SYBR Green dyes If the instrument is not calibrated for the VIC dye the instrument software will be unable to configure the VIC dye layer for the endogenous control gene expression assay Note For more information about the Sequence Detection Systems Spectral Calibration Kit or the calibration procedure see the ABI PRISM 7700 Sequence Detection Systems User Bulletin 4 Generating New Spectra Components P N 4306234 User bulletin 4 can be obtained from Applied Biosystems See To Obtain Documents on Demand on page D 7 To run the TaqMan Human Endogenous Control Plate on a sequence detection system instrument you must configure a plate document with the appropriate assay and sample information The TaqMan Human Endogenous Control Plate compares gene expression levels based on the data collected during the PCR run By configuring the plate document with the sample and assay locations the SDS software can collect and organize the florescence data for analysis To configure the PCR plate document Step Action 1 Open the Sequence Detection System SDS software 2 Create a plate document with the following attributes 7700 Plate Document 5700 Plate Document Single Reporter 5
36. e and genomic DNA contamination are common problems in gene expression studies purify your samples accordingly to ensure the best results If possible use spectrophotometric analysis to determine the concentrations of purified total RNA samples before reverse transcription The table below lists the recommended range of initial template quantities for the reverse transcription RT step Initial Template Quantity of total RNA per 100 uL RT reaction Human Total RNA 60 ng 2 ug Guidelines Follow the guidelines below to ensure optimal RT performance Poly At RNA samples are not recommended for endogenous control experiments because most rRNA has been removed from them A100 uL RT reaction efficiently converts a maximum of 2 yg total RNA to cDNA Perform multiple RT reactions in multiple wells if using more than 2 pg total RNA Use only random hexamers to reverse transcribe the total RNA samples for endogenous control gene expression assays Preparing the The following procedure describes the preparation of three different test Reactions samples and a calibrator sample for reverse transcription Scale the recommended volumes accordingly for the number of samples needed using the TaqMan Reverse Transcription Reagents P N N808 0234 CHEMICAL HAZARD TaqMan Reverse Transcription Reagents may cause eye and skin irritation Always use adequate ventilation such as that provided by a fume hood Please read the MSDS
37. e cDNA specific and their performance was tested using cDNA prepared from human total RNA samples Instruments This protocol describes how to evaluate candidate control gene expression in total RNA samples using the plate and the following 1 2 Introduction sequence detection systems ABI PRISM 7700 Sequence Detection System GeneAmp 5700 Sequence Detection System About TaqMan Endogenous Control Assays About the Internal Positive Control Product Guidelines With the exception of 18S rRNA all assays present on the TaqMan Human Endogenous Control Plate are cDNA specific Each assay has been experimentally proven not to detect up to 10 000 copies of contaminating DNA The 18S rRNA assay is not cCDNA specific However because of the extremely high expression level of rRNA amplification from contaminant DNA has a negligible effect on gene expression values obtained from the plate In spite of these design characteristics Applied Biosystems recommends using only purified total RNA samples Applied Biosystems designed the TaqMan Internal Positive Control IPC to help interpret negative results caused by PCR inhibitors In the absence of inhibitors IPC is co amplified with the target DNA and gives a consistent signal If inhibitors are present the signal generated by the IPC assay diminishes or becomes nonexistent The IPC sequence is artificial to prevent nonspecific amplification Read the following informa
38. e format Data Analysis 4 3 Procedure for Setting the Baseline for the ABI PRISM 7700 Instrument To set the baseline Step Action 1 Identify the components of the linear scale amplification plot as shown on page 4 2 Click the Stop text field in the Baseline box Click here Following the guidelines from the previous page choose from one of the following actions If the amplification plot looks like Then the amplification curve begins after the maximum baseline Do not adjust the baseline the maximum baseline is set too high Decrease the Stop baseline value the maximum baseline is set too low Increase the Stop baseline value Click Update Calculations The SDS software updates the C and standard deviation values 4 4 Data Analysis Setting the Baseline for the GeneAmp 5700 Instrument To set the baseline for the GeneAmp 5700 instrument Step Action 1 In the Plate window select all wells for analysis 2 Select Analysis gt Analyze An SDS warning message appears Click OK to continue In the Plate window click the Results tab Note The tabs just above the wells in the Plate window let you toggle between the Setup Instrument and Results views In the Results view click the Amp Plot tab The Amplification Plot window appears Identify the components
39. ectral noise Guidelines for Setting the Threshold Setting Multiple Thresholds Note Correct placement of the threshold is a crucial step in data analysis Follow the guidelines below to ensure the threshold is set properly To obtain accurate results Setthe threshold value within the exponential phase of the logarithmic scale amplification plots The exponential phase occurs within the range of data points that increase linearly when graphed Set the threshold value so that it is within the exponential phase of all amplification plots If a single threshold cannot be set to satisfy all plots then it must be set multiple times Because the expression levels and AR values of TaqMan endogenous control assays can vary significantly it may be necessary to set the threshold more than once to obtain accurate results If a single threshold value does not intersect the exponential phase of all amplification plots the data must be analyzed and subsequently exported with multiple threshold values The figure below shows a 5700 amplification plot where the threshold must be set independently for each group of curves As shown Threshold 1 is within the exponential phase of the plots in Group A however it intersects with the plateau phase of the plots in Group B The results from this setting would be accurate for the plots in Group A but invalid for the plots in Group B If reset for Group B Threshold 2 the threshold i
40. erase activation Note See your sequence detection systems user s manual for help on setting thermal cycling conditions 3 4 PCR Preparing and Running the PCR Reactions PCR Guidelines Performing PCR The following guidelines ensure optimal PCR performance Do not remove the TaqMan Human Endogenous Control Plate from its foil packaging until you are ready to load the PCR reaction mix Excessive exposure to light can damage the florescent probes Prior to use thaw frozen cDNA samples by placing them on ice When thawed vortex and briefly centrifuge the contents of each tube to resuspend the samples Prepare PCR reaction mixture for each sample in separate microcentrifuge tube before aliquoting it to the reaction plate for thermal cycling and fluorescence analysis The volume of the PCR reaction mix per well must be 50 uL minus the volume of the cDNA sample from the RT step Do not mix the PCR mixture and cDNA samples in the MicroAmp Optical 96 Well Reaction Plate Note The cDNA amplification reaction is optimized with TaqMan Universal PCR Master Mix CHEMICAL HAZARD TaqMan Universal PCR Master Mix may cause eye and skin irritation It may cause discomfort if swallowed or inhaled Always use adequate ventilation such as that provided by a fume hood Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves PCR 3 5 3 6 PCR To perform the P
41. es in efficiency of sample extraction or complementary DNA cDNA synthesis by reverse transcriptase reference A passive or active signal used to normalize experimental results Endogenous and exogenous controls are examples of active references Active reference means the signal is generated as the result of PCR amplification The active reference has its own set of primers and probe R The R value of a reaction containing all components including the template R The R value of an unreacted sample This value can be obtained from the early cycles of a Real Time run the cycles prior to a detectable increase in fluorescence or from a reaction not containing template AR The difference between the R value and the R value It reliably indicates the magnitude of the signal generated by the given set of PCR conditions threshold cycle Cy The value is the cycle at which a statistically significant increase in AR is first detected Calculated as the average standard deviation of R for the early cycles multiplied by an adjustable factor Glossary 1 P N 4308134 Rev D
42. es of cell motility Cyclophilin Cyclophilin is a major cellular component comprising 0 1 0 4 of total cellular huCYC protein It is found in all cells of wide phylogenetic distribution Koletsky et al 1986 It was originally isolated as the main cyclosporin A binding protein Glyceraldehyde GAPDH is a key enzyme involved in glycolysis and is moderately abundant 3 phosphate Allen et al 1987 Its expression changes with insulin treatment and shows dehydrogenase fluctuation through cell cycles and among different cell lines and tissue types huGAPDH Phosphoglycero PGK is a key enzyme involved in glycolysis following GAPDH Because typical kinase huPGK concentrations of glycolytic intermediates are 1 uM for 1 3 bisphosphoglycerate and 118 uM for 3 phosphoglycerate the regulation may be different Bo Microglobulin huB2m B microglobulin is involved with immune response It is moderately abundant and expressed in most tissue types GUssow et al 1987 The level of Bo microglobulin expression may vary in different tissues Okubo et al 1997 B Glucronidase huGUS B glucronidase is a relatively abundant glycoprotein that is expressed constitutively in many tissues It acts as an exoglycosidase in lysomes Oshima et al 1988 Hypoxanthine ribosyl transferase hUHPRT Hypoxanthine ribosyl transferase is located on the X chromosome and is constitutively expressed at low levels Patel ef a 1986 It
43. evels Stable expression provides a reliable basis for comparison with other genes Good Endogenous Control Candidates From the AC profile shown below the 18S ribosomal RNA 18S and transferrin receptor huTfR genes are good candidate controls because their expression remains relatively consistent across the test samples Both assays produced AC values that deviate little from zero indicating a fairly stable level of gene expression relative to the other candidate controls Poor Endogenous Control Candidates In contrast to the 18S and huTfR controls the TATA binding protein huTBP and B Glucronidase huGUS genes are the least desirable choices from the profile in the figure below The expression of both controls vary widely exhibiting AC values that fluctuate in excess of 4 cycles this represents a 16 fold difference in gene expression Calculating Relative Quantification 5 19 2 00 IPC 18S huPO huBA huCYC huGAPDH huPGK huB2m huGUS huHPRT huTBP huTfR 5 5 1 001 ae S 5 Sa 2 e 0 00 L amp S S gt I 3 A iii 5 B25 G 2 A 7 ee EI 3 2 S 5 200 vie 7 a Go gt i 8 E 3 o T E gx 3 00 Bo o N N q fha 2 j bE qi mS q ai Q 400 a y Vai a 2 5 00 X 5 ki BAct Sample 1 WACt Sample 2 ACt Sample 3 Demonstrating TaqMan Human Control Total RNA is available
44. i UAE zmo ZAG O1 mie UAE 0 05 2570 01 Tol Liith ETH 2 8iT fi ary LACH LE 03 ate Ol em Rl URE LESE 03 ZTE Pin UAH 1ESr 05 2610 01 Ias Select Edit gt Copy 5 From the Window menu select the new spreadsheet The new spreadsheet file reappears 6 Click cell A2 7 Select Edit gt Paste Excel pastes the data into the new spreadsheet Calculating Relative Quantification 5 5 To construct a C table continued Step Action 8 Type the labels for the C table as specified in the following table Click on cell Type Al Column B1 Ct Calibrator C1 Ct Calibrator D1 Ct Sample 1 E1 Ct Sample 1 F1 Ct Sample 2 G1 Ct Sample 2 H1 Ct Sample 3 l1 Ct Sample 3 The AC table appears as shown below ee esereer Cs Seely E JEL Me CU 1 eS L 5 6 Calculating Relative Quantification Importing Data to Note This section also consolidates the data from additional files created in the Cy Table the sections Setting Multiple Thresholds on page 4 7 Appendix A Troubleshooting Early Amplification To transfer data from the results file to the C table Step Action 1 From the Window menu select the exported results file The endogenous control plate results spreadsheet reappears From the results file soreadsheet select If viewing a Select cells 5700 results file D2 D13 7700 results file F2 F13
45. ifuge the tubes to force the solution to the bottom of the tube and eliminate air bubbles from the mixture 12 Transfer each reaction to MicroAmp Optical Tube s or Wells of a MicroAmp Optical 96 Well Reaction Plate 13 Cap the MicroAmp Optical tubes or plate 14 Centrifuge the plate or tubes to spin down the contents and eliminate air bubbles from the solutions To perform the RT reaction continued Step Action 15 Load the reactions into a thermal cycler 16 Program your thermal cycler with the following conditions IMPORTANT perform 100 uL reactions If using a 9700 thermal cycler select MAX Mode to Step Incubation Reverse Transcription Reverse Transcriptase Inactivation HOLD HOLD HOLD Temperature 25 0 Ca 48 0 C 95 0 C Time 10 min 30 min 5 min Volume 100 uL a If using random hexamers or oligo d T primers for first strand CDNA synthesis a primer incubation step 25 C for 10 min is necessary to maximize primer RNA template binding Note The thermal cycling parameters are optimal for the Applied Biosystems thermal cyclers listed in Instruments for Reverse Transcription on page 2 4 Due to differences in ramp rates and thermal accuracy you may need to adjust the settings if using another thermal cycler Note See your thermal cycler user s manual for help on setting thermal cycling conditions
46. igned the TaqMan Exogenous Internal Positive Control IPC to help interpret negative results caused by PCR inhibitors In the absence of inhibitors IPC co amplifies with target DNA and gives a specific signal The IPC sequence is artificial so that PCR primers do not amplify anything in the test samples 18S rRNA 18S ribosomal RNA makes up 80 of total RNA and its level is a good indicator for the relative amount of total RNA It is transcribed by a different polymerase from mRNAs and its level is less likely to fluctuate with the test sample The 18S rRNA endogenous reference is the most abundant target on the TaqMan Human Endogenous Control Plate About These Assays B 1 Endogenous Control Role Acidic ribosomal protein huPO Acidic ribosomal protein is moderately abundant Rich et al 1987 and found in most tissue types Because huPO gene expression level seems to remain relatively constant Okubo et al 1997 some researchers select it as their standard when studying samples that are affected by estrogen treatment Beta actin The beta actin gene is ubiquitously expressed in all eukaryotic cells and one of huBA the most frequently used as an internal standard It is a moderately abundant gene constituting 0 1 of mRNA and 0 003 of total RNA Its level fluctuates in some cells and tissues Greenberg et al 1985 Dodge et al 1990 Actins are highly conserved proteins involved in various typ
47. in the starter pack ABI PRISM Optical Adhesive Cover Starter Pack Applied Biosystems P N 4313663 20 optical adhesive covers 1 applicator 1 compression pad Sequence Detection Systems Spectral Calibration Kit Applied Biosystems P N 4305822 TaqMan Reverse Transcription Reagents Applied Biosystems P N N808 0234 Centrifuge with 96 well plate adapter MLS Disposable gloves MLS Microcentrifuge MLS Microcentrifuge tubes sterile 1 5 mL MLS MicroAmp Reaction Tubes with Caps Applied Biosystems P N N802 0540 Microsoft Excel or equivalent software Software vendors Introduction 1 11 1 12 Introduction User supplied materials continued Materials Source Pipette tips aerosol resistant MLS Pipettors MLS Positive displacement Air displacement Polypropylene tubes MLS Water RNase free distilled deionized MLS a Only for 7700 instruments not calibrated with the VIC dye See Configuring the ABI Prism 7700 Software for the VIC Dye on page 3 2 for more information Safety Documentation User Attention Words Chemical Hazard Warning Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for pro
48. later during spreadsheet analysis To set multiple thresholds continued Step Action 6 Follow the procedure for spreadsheet analysis as described in Calculating the Relative Quantification Using a Spreadsheet on page 5 5 Setting the Changing the Y Axis to Logarithmic Scale Threshold Value l for the ABI PRISM To view the threshold value 7700 Instrument Step Action 1 Double click the AR label on the Y axis of the graph The Scale dialog box appears 2 Click the Logarithmic Scale radio button from the Display box Baa play Gh Lieve Beale L Err eer L Click here 3 Click OK The amplification plot appears in logarithmic format Procedure for Setting the Baseline for the ABI PRISM 7700 Instrument To set the threshold value Step Action 1 Identify the components of the amplification curve as shown in Threshold Value Basics on page 4 6 2 Click and drag the threshold line so that it is Above the background noise Below the plateaued region Within the exponential phase of the amplification curve Data Analysis 4 9 To set the threshold value continued Step Action Below the plateaued region Within this range Above the background a ia FITE Hiiik i a H RH HHH Note To reset the threshold to the default value click the Suggest button in the Threshold b
49. m enter the requested information and your question then click Ask Us RIGHT NOW Within 24 to 48 hours you will receive an e mail reply to your question from an Applied Biosystems technical expert To Obtain Free 24 hour access to Applied Biosystems technical documents Documents on including MSDSs is available by fax or e mail or by download from our Demand Web site To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index number c Use the index number when requesting documents following the procedures below by phone for fax delivery a From the U S or Canada call 1 800 487 6809 or from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request Technical Support D 7 To Obtain Customer Training Information D 8 Technical Support To order documents Then through the a Access the Applied Biosystems Technical Support Web Internet for fax site at or e mail http www appliedbiosystems com techsupp delivery b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed for
50. m then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax deliver but no limit on the number of documents you can order for e mail delivery The Applied Biosystems Training web site at www appliedbiosystems com techsupp training html provides course descriptions schedules and other training related information Glossary calibrator A sample used as a basis for comparison with the other samples on the TaqMan Human Endogenous Control Plate endogenous control RNA or DNA that is present in each experimental sample as isolated By using an endogenous control as an active reference you can normalize quantification of a messenger RNA mRNA target for differences in the amount of total RNA added to each reaction exogenous control Characterized RNA or DNA spiked into each sample at a known concentration An exogenous active reference is usually an in vitro construct that can be used as an internal positive control IPC to distinguish true target negatives from PCR inhibition An exogenous reference can also be used to normalize for differenc
51. mple on the TaqMan Human Endogenous Control Plate The figure below illustrates the recommended plate configuration OOOO 0000000 FOOOOOOOOOOOO OOOO OOOCOOOCO 2 OOOO OOCeCee i COCOON O I ae FOOOOOOO0CO00O IID COOOOOOCOO ana MOOCCOOOOOOC The calibrator sample serves the following purposes Provides a baseline for comparison with the other samples on the plate Serves as a basis for comparing sample data from multiple independently run plates Note The calibrator sample can be used to compare sample data from independently run plates only if the same calibrator sample is present on all plates Reverse Transcription 2 3 Reverse Transcription for All Amplicons Except 18S Reverse The following guidelines ensure optimal RT performance Transcription Guidelines Instruments for Reverse Transcription 2 4 Reverse Transcription Depending on gene expression levels in your samples Applied Biosystems recommends using 10 100 ng of total RNA converted to cDNA per well Enough sample specific CDNA must be generated for each sample to fill 24 wells on the TaqMan Human Endogenous Control Plate Perform multiple RT reactions in multiple wells if using more than 2 ug total RNA A maximum of 2 yg total RNA per 100 uL RT reaction efficiently converts to cDNA Prior to use thaw all reagents except the enzyme and the RNase Inhibitor When the reagents are thawed keep them on ice Keep the
52. nation Poly At The 18S rRNA assay cannot accurately evaluate poly A RNA samples because most of the rRNA has been removed Non human Except for the 18S rRNA and the internal positive control IPC all assays on the endogenous control plate are human specific Because the quality of results is directly related to the purity of the RNA template Applied Biosystems recommends using only well purified samples with the TaqMan Human Endogenous Control Plate Because ribonuclease and genomic DNA contamination are common problems in gene expression studies purify your samples accordingly to ensure the best results IMPORTANT Each TaqMan endogenous control assay has been experimentally proven not to detect up to 10 000 copies of contaminating DNA In spite of this design characteristic Applied Biosystems recommends using purified total RNA samples to obtain the best results If possible use spectrophotometric analysis to determine the concentrations of your purified total RNA samples The table below contains the recommended range of template quantity Initial Template Quantity Per Well Human Total RNA 10 100 ng a Initial RNA converted to cDNA IMPORTANT Enough sample specific cDNA must be generated for each sample to fill 24 wells on the TaqMan Human Endogenous Control Plate About the Applied Biosystems recommends evaluating duplicate rows of three Calibrator Sample test samples and a calibrator sa
53. nd produce errors when calculating gene expression In This Chapter The following topics are discussed in this chapter Topic See Page Setting the Baseline 4 2 Setting the Threshold Value 4 6 Data Analysis 4 1 Setting the Baseline Baseline Basics The baseline is a defined range of cycles before the Sequence Detection Systems SDS software detects the amplification of PCR product The SDS software uses a default range of cycles 3 15 on 7700 instruments and cycles 6 15 on 5700 instruments to establish the baseline The figure below illustrates the important characteristics of the baseline on a 7700 amplification plot 3 500 3 000 2 500 2 000 4 500 1 000 ARn 0 500 Initial amplification baseline must end before this point 10 20 30 40 Cycle Product amplification Default 7700 baseline cycles 3 15 Because of the abundance of rRNA low C values can be obtained in TaqMan RT PCR applications with the 18S assays When the amplification of the 18S target reaches a detectable level at a very early cycle it can limit the number of cycles over which the software calculates the baseline In rare cases this interferes with the detection of less abundant targets See Appendix A Troubleshooting Early Amplification for more information Guidelines for Correct placement of the baseline is a crucial step in data analysis Setting the Follow the guidelines below to ensu
54. ned to detect the presence of PCR inhibitors in test samples In This Chapter The following topics are discussed in this chapter Topic See Page Control Plate Assay System 1 2 Preventing Contamination 1 9 Materials and Equipment 1 10 Safety 1 13 Introduction 1 1 Control Plate Assay System Purpose of the Kit Applied Biosystems developed the TaqMan Human Endogenous Control Plate to simplify endogenous control selection by eliminating several major developmental obstacles The following table explains difficulties researchers face when investigating potential controls and how the plate alleviates these problems Obstacle Solution Assay development and optimization is expensive and time consuming The TaqMan Human Endogenous Control Plate features 11 preoptimized ready to use control gene assays Several studies indicate that expression of traditional housekeeping genes such as GAPDH and beta actin varies among tissues and developmental stages Bonini and Hofmann 1991 Spanakis 1993 The TaqMan Human Endogenous Control Plate simultaneously evaluates eleven candidate controls that cover a broad range of biological functions and vary in expression levels Recent studies indicate that pseudogenes and related genes make RT PCR results unreliable unless the PCR primers are cDNA specific Raff et al 1997 Multimer et al 1998 TaqMan endogenous control assays ar
55. ng MSDSs_ You can order free additional copies of MSDSs for chemicals 1 14 Introduction manufactured or distributed by Applied Biosystems using the contact information below To order MSDSs Then Over the Internet a Go to our Web site at www appliedbiosystems com techsupp b Click MSDSs If you have Then The MSDS document number or the Document on Demand index number Enter one of these numbers in the appropriate field on this page The product part number Keyword s Select Click Here then enter the part number or keyword s in the field on this page c You can open and download a PDF using Adobe Acrobat Reader of the document by selecting it or you can choose to have the document sent to you by fax or email By automated telephone service from any country Use To Obtain Documents on Demand on page D 7 By telephone in the United States Dial 1 800 327 3002 then press 1 By telephone from Canada To order in Then dial 1 800 668 6913 and English Press 1 then 2 then 1 again French Press 2 then 2 then 1 By telephone from any other country See the specific region in To Contact Technical Support by Telephone or Fax on page D 3 For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Site Preparation A site preparation an
56. ntersects Group A at a point very early in the exponential phase where background noise causes non reproducibility The solution for this situation is to set the threshold separately for both groups Data Analysis 4 7 4 8 Data Analysis 10 1 Plot Group A Plot Group B i 0 1 Beebe pea peach hepa calle Threshold 1 i valid for Group A CE SRR SREB RTH shee we l aaea Threshold 2 0 01 F valid for Group B Ai LL A AN Fi i i fi oon 0 10 20 30 40 Cycle To set multiple thresholds Step Action 1 Following the appropriate procedure for your instrument set a threshold value that is valid for the majority of plots on the logarithmic graph 2 Export the data as explained in Exporting and Viewing the Results File on page 5 2 The software saves the data to a file 3 From the logarithmic amplification plot identify the plots for which the threshold set in step 1 was invalid 4 Reset the threshold value for the second group of plots 5 Export the data as explained in Exporting and Viewing the Results File on page 5 2 IMPORTANT Save the file with a name different than that used in step 2 The software overwrites files with identical names There are now two files on the disk The file created in step 2 containing valid data for the majority of plots from the experiment The file created in step 5 containing valid data for the remaining plots Note The data in the files is combined
57. nthesis 1 800 899 5858 then press 1 then 5 1 508 383 7855 FMAT 8100 HTS System Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 1 then 6 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 LC MS Applied Biosystems MDS Sciex 1 800 952 4716 1 508 393 7899 Outside North America Telephone Fax Region Dial Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 Africa French Speaking Courtaboeuf Cedex France 33 1 69 59 85 11 33 1 69 59 85 00 South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493 Region Telephone Dial Fax Dial Easter n Asia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 or 86 10 64106617 86 800 8100497 Hong Kong 852 2756 6928 852 2756 6968 India New Delhi 91 11 653 3743 3744 91 11 653 3138 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 79588268 603 79549043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 2358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66
58. ntries not listed 44 0 1925 282481 44 0 1925 282509 Warrington UK Japan Japan Hacchobori Chuo Ku Tokyo 8120 477392 Toll free within Japan or 81 3 5566 6230 8120 477120 Toll free within Japan or 81 3 5566 6507 Latin America Caribbean countries Mexico and Central 52 55 35 3610 52 55 66 2308 America Brazil 0 800 704 9004 or 55 11 5070 9694 95 55 11 5070 9654 Argentina 800 666 0096 55 11 5070 9694 95 Chile 1230 020 9102 55 11 5070 9694 95 Uruguay 0004 055 654 55 11 5070 9694 95 We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products You can also order technical documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms interest then select the relevant support region for the product area of 3 In the Personal Assistance form enter the requested information and your question then click Ask Us RIGHT NOW To submit technical questions from North America or Europe Step Action 4 In the Customer Information for
59. of the linear scale amplification plot as shown in Baseline Basics on page 4 2 Click the Analysis Preferences button or select Edit gt Preferences The Preferences dialog box appears In the Baseline box highlight the current Start and Stop values and type in new values IMPORTANT When selecting a baseline refer to the guidelines listed in Guidelines for Setting the Baseline on page 4 2 Click OK Select Analysis gt Analyze The software performs the analysis The system beeps when the analysis is complete Note For help on setting the baseline see the GeneAmp 5700 Sequence Detection System User s Manual P N 4304472 Data Analysis 4 5 Setting the Threshold Value Threshold Value For the 7700 instrument the default threshold value is the average Basics standard deviation of AR within the defined baseline region multiplied by an adjustable factor The SDS software calculates the threshold value as ten standard deviations from the baseline For this reason the baseline must be set before you adjust the threshold value The threshold value must be set manually for the 5700 instrument 4 6 Data Analysis The figure below illustrates the important characteristics of the threshold on a 7700 plot ARn Characteristic Description 1 Product amplification Plateau phase Exponential phase Threshold value o AeA OIN Background sp
60. on for All Amplicons Except 18S 2 4 Reverse Transcription for the 18S Amplicon 0 0 eee ee 2 8 PCR OVEEVIEW iis cate et wists Greate a entero a poe Tay nN AR EN NAN web 3 1 Preparing the Sequence Detection System for PCR 3 2 Preparing and Running the PCR Reactions 0004 3 5 Data Analysis OVVIE W ome 9 Ok ote etd Oa E gees Dele ATE Dad pune 4 1 Settings the Baseline 2 18 chew Soba E reba euha geehatned 4 2 Setting the Threshold Value 0 00 0 cece eee eee eee 4 6 Calculating Relative Quantification OVELVICW ois ccleaner Dace ai gah is Beet haere ee 5 1 Exporting and Viewing the Results File 0 004 5 2 Calculating the Relative Quantification Using a Spreadsheet 5 5 Interpreting Results 0 0 00 eee eee eee 5 17 Troubleshooting Early Amplification About These Assays References Technical Support Technical SUpport s porroi cele ian he tala eke Via ee Rik ew BFE a BLS D 1 Glossary Introduction Overview About This The TaqMan Human Endogenous Control Plate is a research tool Product designed to simplify the selection of endogenous controls for gene expression studies The plate evaluates the expression of eleven select housekeeping genes in total RNA samples using a two step reverse transcription polymerase chain reaction RT PCR The plate also features a unique internal positive control IPC desig
61. ox Click here 3 Click Update Calculations The SDS software updates the C and standard deviation values 4 Click OK 4 10 Data Analysis Setting the Threshold Value for the GeneAmp 5700 Instrument To set the threshold for the GeneAmp 5700 instrument Step Action 1 In the Plate window click the Results tab Note The tabs just above the wells in the Plate window let you toggle between the Setup Instrument and Results views In the Results view click the Amp Plot tab The Amplification Plot window appears Identify the components of the amplification curve as shown in Threshold Value Basics on page 4 6 Determine a value for threshold that is Above the background noise Below the plateaued region Within the exponential phase of the amplification curve IMPORTANT When selecting a threshold refer to the guidelines listed in Guidelines for Setting the Threshold on page 4 7 Click the Analysis Preferences button or select Edit gt Preferences The Preferences dialog box appears In the Threshold box enter the value you determined in step 4 above Click OK Select Analysis gt Analyze The software performs the analysis The system beeps when the analysis is complete Note For help on setting the threshold value see the GeneAmp 5700 Sequence Detection System User s Manual P N 4304472 Data Analysis
62. per instrument operation 7 er NGREe Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices PNG Indicates a potentially hazardous situation which if not avoided could result in death or serious injury OLANE indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations NTUN CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with and inhalation of chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Do not leave chemical containers open Use only with adequate ventilation Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Introduction 1 13 Orderi
63. rage 5 fle fi i of ira Ff 4 14 be TRE 14h et C for this row and z m iE Li FIR must be deleted How to Delete Invalid Cy Values To delete an invalid C value from the spreadsheet Step Action 1 Click the cell containing an invalid C to select it 2 Select Edit gt Clear gt All 5 10 Calculating Relative Quantification Averaging Before calculating AC values for the calibrator and samples average Duplicate Cy the C values from duplicate wells Because the samples and calibrator Values are arrayed twice across the endogenous control plate the exported data for every sample contains two C values for each target control To calculate AC values you must average the values for these duplicate wells To add Average C columns to your C table Step Action 1 Create columns for the average calibrator and sample C values by inserting new columns into the spreadsheet as follows Click cell Select D1 Select Insert gt Columns inert Teln 1 AES Warkuheert thart F haco fr fanction_ Name F Mabe ject Excel inserts a new column before column D G1 Select Insert gt Columns Excel inserts a new column before column G J1 Select Insert gt Columns Excel inserts a new column before column J 2 Type the labels for the C table as specified in the following table Click on cell Type
64. re the baseline is set properly Baseline Set the baseline so that the initial amplification curve begins at a 4 2 Data Analysis cycle that is greater than the maximum value of the baseline Do not adjust the default baseline if the amplification curve growth begins after cycle 15 For example the default can be used for the plot above because initial amplification occurs at cycle 16 Setting the Before setting the baseline you must first Baseline for the 4 Display the results on an amplification plot ABI PRISM 7700 Instrument Change the Y axis to linear scale Displaying Results on an Amplification Plot To display the results on an amplification plot Step Action 1 Select Analysis gt Analyze The SDS software analyzes the raw data and displays an amplification plot 2 If the SDS software does not display an Amplification Plot then select Analysis gt Amplification Plot The SDS software displays an amplification plot log AR vs Cycle Changing the Y Axis to Linear Scale To change the Y axis to linear scale Step Action 1 Double click the AR label on the Y axis of the amplification plot oe Double click here T a oi ral Ti eee The Scale dialog box appears 2 Click the Linear Scale radio button to graph the data on a linear scale pe Click here EJ Lime Fede E ogiri beil 3 Click OK The amplification plot appears in a linear scal
65. spreadsheet application To view the exported results file Step Action 1 Open the spreadsheet software 2 Select File gt Open 3 Select from one of the following If you created Then select the one results file exported results file and click Open two results files as explained in the Setting Multiple Thresholds on page 4 7 exported file created in steps 1 2 and click Open two results files as explained in How to Correct for Early Amplification on page A 2 exported file created in steps 1 4 and click Open 5 4 Calculating Relative Quantification Calculating the Relative Quantification Using a Spreadsheet Overview Constructing a Cr Table Applied Biosystems recommends using a spreadsheet to create comparative gene expression profiles from TaqMan Human Endogenous Control Plate data To construct a C table Step Action 1 Select File gt New A new spreadsheet appears 2 From the Window menu select the results file PA eet ay Sew Winrar Arangi Heme iplit Freene Panes Shire Nipheard iAarkiaak Pate results The endogenous control plate results spreadsheet reappears 3 Select cells A2 A13 E o E F Te Diera Hi h in Li Lith 2 50 05 i E i T Ja UAH Late Os 1 Bate Ol TE URE 2230 05 1E 0I Ta UR EH 1 7ar 05 Z010 O1 mola Liith 7 B 05 di Qi oa ay LACH 450 03 C Ot Fa
66. sults File on page 5 2 The software saves the data The results in the file are valid only for the wells that amplify during the very early cycles You now have two results files A file containing valid data for plots appearing in the later cycles A file containing valid data for plots appearing in the early cycles Follow the procedure for spreadsheet analysis as described in Calculating the Relative Quantification Using a Spreadsheet on page 5 5 Troubleshooting Early Amplification A 3 About These Assays Overview The TaqMan Human Endogenous Control Plate evaluates the expression of eleven common housekeeping genes and an internal positive control in total RNA samples Applied Biosystems designed TaqMan assay primers and probes to be cDNA specific to avoid problems associated with pseudogenes related genes and contaminant genomic DNA Quality Control Applied Biosystems tests the preloaded primers and probes on the TaqMan Human Endogenous Control Plate as part of a manufacturing quality control process In this process the performance of each endogenous control target was gauged using cDNA prepared from human total RNA samples Each assay demonstrated that it did not detect up to 10 000 copies of contaminating genomic DNA Description of The following table lists the potential controls and their cellular Endogenous functions Controls Endogenous Control Role IPC Applied Biosystems des
67. t tt ba THE 12 hy TH ee fet ee ee aie E E Calculating Relative Quantification 5 15 To calculate AC values for the calibrator and samples continued Step Action Select and copy cells C17 G17 Excel changes the boarder of the selected cell to a dotted line indicating that the cell is ready for duplication Select cells F18 G28 and paste the selection into the spreadsheet Tae pel 1 2 mE a 4 hu Be 3 hulle Shei POH 7 hu Pit buble T he GUS 10 huHPet ii bs THE 12 hy TH Excel automatically copies the formulas in cells C17 G17 to the cells below AAR hele Ai 5 16 Calculating Relative Quantification Interpreting Results Overview To interpret the results from the spreadsheet analysis create a profile of control gene expression from the data in the AC table Interpreting results consists of the following steps Topic See Page Generating a Gene Expression Profile 5 17 Interpreting the Gene Expression Profile 5 18 The Relationship Between AC and Gene Expression 5 18 Choosing an Endogenous Control 5 19 Demonstrating Performance with TaqMan Human Control Total 5 20 RNA Generating a Gene The following procedure describes how to generate a profile using the Expression Profile Excel Chart Wizard To graph your results using the Excel Chart Wizard Step Action 1 Select cells A16 E28 A TT SL ES
68. t PCR and Time Release PCR into existing amplification systems with little or no modification of cycling parameters or reaction conditions These techniques improve amplification of most templates by lowering background and increasing amplification of specific products TaqMan Universal PCR Master Mix is 2X in concentration and contains sufficient reagent to perform 200 reactions 50 uL each The mix is optimized for TaqMan reactions and contains AmpliTaq Gold DNA Polymerase AmpErase UNG dNTPs with dUTP Passive Reference and optimized buffer components Preventing Contamination Introduction About AmpErase UNG General PCR Practices Because of the high throughput and repetitive nature of the 5 nuclease assay special laboratory practices are necessary in order to avoid false positive amplifications Kwok and Higuchi 1989 This is because of the capability for single DNA molecule amplification provided by the PCR process Saiki et al 1985 Mullis and Faloona 1987 AmpErase uracil N glycosylase UNG is a pure nuclease free 26 kDa recombinant enzyme encoded by the Escherichia coli uracil N glycosylase gene This gene was inserted into an E coli host to direct expression of the native form of the enzyme Kwok and Higuchi 1989 UNG acts on single and double stranded dU containing DNA It acts by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The enzyme causes the release of uracil thereby creating an
69. t with the recommendd values above c Perform multiple RT reactions in multiple wells if using more than 2 ug total RNA Note The calibrator is a sample used as a basis for comparison with the other samples on the plate see About the Calibrator Sample on page 2 3 for more information 2 Label four 1 5 mL microcentrifuge tubes for three test samples and a calibrator sample 3 Transfer 60 ng to 2 pg up to 38 5 uL of each total RNA sample to the corresponding microcentrifuge tube Reverse Transcription 2 5 2 6 Reverse Transcription To perform the RT reaction continued Step Action 4 If necessary dilute each total RNA sample to a volume of 38 5 uL with RNase free deionized water 5 Cap the tubes and gently tap each to mix the diluted samples 6 Briefly centrifuge the tubes to eliminate air bubbles in the mixture 7 Label four 0 2 mL MicroAmp Reaction tubes for the three total RNA samples and a calibrator sample 8 Pipette 61 5 uL of reaction mix from step 1 into each MicroAmp Reaction Tube from step 7 10X RT buffer gt e MgCl __ dNTPs mixture Random hexamers e MultiScribe reverse transcriptase _ RNase inhibitor 61 5 uL 61 5 uL 61 5 uL 61 5 uL Calibrator Sample 1 Sample 2 Sample 3 9 Transfer 38 5 uL of each dilute total RNA sample to the corresponding MicroAmp Reaction tube 10 Cap the reaction tubes and gently mix the reactions 11 Breifly centr
70. tion before proceeding The endogenous control plate cannot be used to conduct multiplex experiments It is designed only as a tool to aid in the selection of endogenous controls The endogenous control plate should not be used to assay poly At RNA samples The 18S rRNA assay cannot evaluate poly At RNA samples because most of the ribosomal RNA has been removed Applied Biosystems designed the plate to evaluate only total RNA Reverse transcription of total RNA to cDNA must be done using random hexamers ABI PRISM 7700 Sequence Detection Systems must be calibrated for the VIC dye before running the TaqMan Human Endogenous Control Plate See Configuring the ABI Prism 7700 Software for the VIC Dye on page 3 2 for more information The endogenous control plate is optimal for use with the following ABI PRISM 7700 Sequence Detection System and GeneAmp 5700 Sequence Detection System TaqMan Universal PCR Master Mix P N 4304437 TaqMan Reverse Transcription Reagents P N N808 0234 Introduction 1 3 About the TaqMan Human Endogenous Control Plate 1 4 Introduction The TaqMan Human Endogenous Control Plate is a MicroAmp Optical 96 Well Reaction Plate divided into 12 columns one for every control assay Each column consists of eight identical wells containing TaqMan primers and probes for the detection of one target gene The figure below illustrates the assay configurations on the plate
71. xpression assay Step Action 1 Select Analysis gt Export gt Ct The Save As dialog box appears Note You can also click the Export button in the Report window to open the Save As dialog box Click the Save as text box and type a name for the results file Click Save The SDS software exports the data to a comma separated text file 4 Close the SDS software The figure below is an example of an exported 5700 results file as viewed with the Microsoft Excel spreadsheet 5 2 Calculating Relative Quantification Exporting Results from a ABI PRISM 7700 Sequence Detection System To export the data from the endogenous control gene expression assay Step Action 1 Select File gt Export gt Results 2 Click the Export result data as text box and type a name for the file 3 Click the Export All Wells radio button Export result daba as Click here ab Export AN Wells Q Export Setected Wells The software saves the data from all wells to the results file 4 Click Export The SDS software exports the data to a Microsoft Excel spreadsheet 5 Close the SDS software The figure below is an example of an exported 7700 results file as viewed with the Microsoft Excel spreadsheet Calculating Relative Quantification 5 3 Viewing the The exported SDS file from the data analysis procedure can be viewed Results File using almost any

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TaqMan® Human Endogenous Control Plate