TaqMan Cytokine Gene Expression Plate I Protocol

Transcription

1 TaqMan Cytokine Gene Expression Plate I Protocol For Research Use Only. Not for use in diagnostic procedures.

2 Copyright 2003, Applied Biosystems For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. NOTICE TO PURCHASER: DISCLAIMER OF LICENSE The TaqMan Cytokine Gene Expression Plate I (P/N ) is optimized for use in the Polymerase Chain Reaction ( PCR ) and 5 nuclease detection methods covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No license under these patents to use the PCR process or 5 nuclease detection methods is conveyed expressly or by implication to the purchaser by the purchase of this product. A license to use the PCR process for certain research and development activities accompanies the purchase of certain Applied Biosystems reagents when used in conjunction with an authorized thermal cycler, or is available from the Applied Biosystems. Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, or at Roche Molecular Systems, Inc.,1145 Atlantic Avenue, Alameda, California ABI PRISM and its design, and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. Applied Biosystems, MultiScribe, and VIC are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. AmpErase, AmpliTaq Gold, 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. Part Number Rev. C 05/2003

3 Contents 1 Introduction Overview Cytokine Plate Design and Assay System Preventing Contamination Materials and Equipment Safety Reverse Transcription Overview Preparing the RNA Template Creating an RT Plate Document Performing Reverse Transcription PCR Overview Creating and Configuring a Cytokine Plate Document Setting Thermal Cycling Conditions Preparing and Running the PCR Reaction Data Analysis Overview Before the Analysis Setting the Baseline Values Setting the Baseline for the FAM Dye Layer Setting the Threshold Value for the FAM Dye Layer iii

4 Setting the Baseline for the VIC Dye Layer Setting the Threshold Value for the VIC Dye Layer Calculating Relative Quantification Overview Exporting and Viewing the Results File Analyzing Multiplex PCR Results (Comparative C T Method) Manually Calculating Relative Quantification Values Calculating the Relative Quantification Using a Spreadsheet Interpreting Results A Background Information B Cytokine Information C References D Technical Support Glossary iv

5 Introduction 1 1 Overview About This Product In This Chapter Applied Biosystems developed the TaqMan Cytokine Gene Expression Plate I (P/N ) as a research tool for real-time, in vitro quantitative evaluation of human cytokine gene expression. The plate detects the expression of 12 cytokine target sequences and an endogenous control in complementary DNA (cdna) samples. The procedure outlined in this protocol describes how to evaluate cytokine gene expression using this plate and the ABI PRISM 7700 Sequence Detection System (7700 SDS). The following topics are discussed in this chapter: Topic See Page Cytokine Plate Design and Assay System 1-2 Preventing Contamination 1-5 Materials and Equipment 1-6 Safety 1-8 Introduction 1-1

6 Cytokine Plate Design and Assay System Cytokine Gene Expression Plate I Setup The TaqMan Cytokine Gene Expression Plate I consists of a MicroAmp Optical 96-Well Reaction Plate arranged into 12 columns, one for every cytokine in the assay. Each column is made up of eight identical wells containing TaqMan primers and probes for the assay of one human cytokine mrna and an 18S endogenous control. The figure below illustrates the primer and probe configurations for the cytokine gene expression plate A IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα B IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα C IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα D IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα E IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα F IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα G IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα H IL-α IL-1β IL-2 IL-4 IL-5 IL-8 IL-10 IL-12p35 IL-12p40 IL-15 IFNγ TNFα Each well on the cytokine plate contains dried TaqMan primers and probes for the detection of one cytokine cdna target and an endogenous control. The TaqMan Cytokine Gene Expression Plate I uses two dye layers to detect the presence of the target and control sequences. The figure below illustrates the dye layer configurations. FAM layer cytokine targets VIC layer endogenous control The FAM dye layer yields the results for quantification of the cytokine target mrna, while the VIC dye layer yields the results for quantification of the 18S ribosomal RNA endogenous control. 1-2 Introduction

7 Guidelines Although the TaqMan Cytokine Gene Expression Plate I is designed for the general assay of total RNA samples, the plate does have some restrictions for use. Please consider the following information before proceeding: The TaqMan Cytokine Gene Expression Plate I should not be used to assay poly A + RNA samples, because most of the rrna has been removed from them. The plate is designed to assay only total RNA. Reverse transcription of total RNA to cdna for cytokine gene expression assays must be done with random hexamers. The 7700 SDS must be calibrated with the Sequence Detection Systems Spectral Calibration Kit (P/N ) before performing assays with the TaqMan Cytokine Gene Expression Plate I. See ABI PRISM 7700 Sequence Detection System User Bulletin #4: Generating New Spectra Components (P/N ) for the spectral calibration kit procedure. The TaqMan Cytokine Gene Expression Plate I is optimized for use with the following Applied Biosystems products: ABI PRISM 7700 Sequence Detection System TaqMan Universal PCR Master Mix (P/N ) TaqMan Reverse Transcription Reagents (P/N N ) Master Mix Description The TaqMan Universal PCR Master Mix is 2X in concentration and contains sufficient reagent to perform 200 reactions (50 µl 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. Introduction 1-3

8 Cytokine Gene Expression Assay System RNA quantification assays in the TaqMan Cytokine Gene Expression Plate I are performed in a two-step reverse transcription polymerase chain reaction (RT-PCR). The figure below illustrates the assay steps. RT Step Extension of primer on mrna 5' 3' Synthesis of 1st cdna strand 5' Random Hexamer 3' 5' cdna cdna mrna PCR Step Extension of primer on cdna 3' 5' 5' Forward Primer Synthesis of 2nd cdna strand 3' 5' 5' 3' PCR amplification of cdna Forward Primer 5' 3' 5' 5' 3' Reverse Primer 5' Cycle #1 Cycle #2 GR1312b In the first step, cdna is reverse transcribed from total RNA samples using random hexamers from the TaqMan Reverse Transcription Reagents (P/N N ). In the second step, PCR products are synthesized from cdna samples using the TaqMan Universal PCR Master Mix (P/N ). About DNA Contamination IMPORTANT The 12 cytokine assays present on the TaqMan Cytokine Gene Expression Plate I have been designed to be RNA specific. Each assay has been demonstrated not to detect up to 10,000 copies of contaminating DNA. The endogenous 18S rrna assay included in all wells of the TaqMan Cytokine Gene Expression Plate I is not RNA specific. However, due to the extremely high expression level of rrna, only gross DNA contamination can cause errors in relative quantification values obtained from the TaqMan Cytokine Gene Expression Plate I. In spite of these design characteristics, Applied Biosystems still recommends using purified total RNA samples to obtain the best performance when using the TaqMan Cytokine Gene Expression Plate I. 1-4 Introduction

9 Preventing Contamination Introduction AmpErase UNG Due to 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 et al., 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 has been 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 alkali-sensitive apyrimidic site in the DNA. The enzyme has no activity on RNA or dt-containing DNA (Longo et al., 1990). General PCR Practices Please follow these recommended procedures: Wear a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation) and clean gloves when preparing samples for PCR amplification. Change gloves whenever you suspect that they are contaminated. Maintain separate areas and 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 a positive-displacement pipette or aerosol-resistant pipette tips. Clean lab benches and equipment periodically with 10% bleach solution. Introduction 1-5

10 Materials and Equipment Kit Components TaqMan Cytokine Gene Expression Plate I is available in two configurations: P/N Contents TaqMan Cytokine Gene Expression Plate I (P/N ) a TaqMan Universal PCR Master Mix (P/N ) TaqMan Control Total RNA (Human) (P/N ) TaqMan Cytokine Gene Expression Plate I (P/N ) a TaqMan Universal PCR Master Mix (P/N ) a. Contains two MicroAmp Optical 96-Well Reaction Plates and Optical Caps Materials Items required for cytokine gene expression assays: Equipment/Software ABI PRISM 7700 Sequence Detection System TaqMan Universal PCR Master Mix TaqMan Control Total RNA (Human) TaqMan Reverse Transcription Reagents Sequence Detection Systems Spectral Calibration Kit Note Source Applied Biosystems Applied Biosystems (P/N ) Applied Biosystems (P/N ) Applied Biosystems (P/N N ) Applied Biosystems (P/N ) The MicroAmp Optical 96-Well Reaction Plate may be sealed with MicroAmp Optical Caps, or ABI PRISM Optical Adhesive Cover MicroAmp Optical 96-Well Reaction Plate/Optical Caps ABI PRISM Optical Adhesive Cover Starter Pack 20 optical adhesive covers 1 applicator 1 compression pad Applied Biosystems (P/N ) Applied Biosystems (P/N ) 1-6 Introduction

11 Items required for cytokine gene expression assays: (continued) Equipment/Software MicroAmp Reaction Tubes with Caps Microsoft Excel Microcentrifuge Centrifuge with 96-well plate adapter Polypropylene tubes Pipettors, positive-displacement or air-displacement Pipette tips RNase-free water Disposable gloves Source Applied Biosystems (P/N N ) Major software vendor Major laboratory supplier (MLS) MLS MLS MLS MLS MLS MLS Storage and Stability Upon receipt store the TaqMan Cytokine Gene Expression Plate I, at 2 8 C. Introduction 1-7

12 Safety Documentation User Attention Words 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 operation. Indicates information that is necessary for proper instrument! CAUTION 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.! WARNING Indicates a potentially hazardous situation which, if not avoided, could result in death or serious injury.! DANGER 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. Chemical Hazard Warning! WARNING 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. 1-8 Introduction

13 Site Preparation and Safety Guide A site preparation and safety guide is a separate document sent to all 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. Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below. To order MSDSs... Over the Internet Then... a. Go to our Web site at b. Click MSDSs. If you have... The MSDS document number or the Document on Demand index number The product part number Keyword(s) Then... Enter one of these numbers in the appropriate field on this page Select Click Here, then enter the part number or keyword(s) in the field on this page. By automated telephone service from any country By telephone in the United States By telephone from Canada By telephone from any other country 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 . Use To Obtain Documents on Demand on page D-7. Dial , then press 1. To order in... Then dial and... English Press 1, then 2, then 1 again French Press 2, then 2, then 1 See To Contact Technical Support by Telephone or Fax on page D-3. Introduction 1-9

14 1-10 Introduction For chemicals not manufactured or distributed by Applied Biosystems, call the chemical manufacturer.

15 Reverse Transcription2 2 Overview About This Chapter In This Chapter This chapter covers reverse transcription (RT), a process in which cdna is synthesized from total RNA samples. Reverse transcription is the first step in the two-step RT-PCR cytokine gene expression quantification experiment, as described in Cytokine Gene Expression Assay System on page 1-4. In this step, random hexamers from the TaqMan Reverse Transcription Reagents prime total RNA samples for reverse transcription using MultiScribe Reverse Transcriptase. The following topics are discussed in this chapter: Topic See Page Preparing the RNA Template 2-2 Creating an RT Plate Document 2-3 Performing Reverse Transcription 2-4 Reverse Transcription 2-1

16 Preparing the RNA Template Recommended Template Template Quality Template Quantity Use only total RNA samples to generate cdna for use with the TaqMan Cytokine Gene Expression Plate I. 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. The quality of the results is directly related to the purity of the RNA template used. Therefore, use only well-purified samples with the Cytokine Gene Expression Plate I. Because ribonuclease 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-µL RT reaction) Total RNA 60 ng 2 µg 2-2 Reverse Transcription

17 Creating an RT Plate Document Opening a New Plate Document If you are performing the RT step on a 7700 SDS, you must first construct a plate document as described below. If you are using another thermal cycler for this step, skip the procedure below and go to the next page. To set up a plate document on the 7700 SDS: Step Action 1 Open the 7700 SDS software. The 7700 SDS software opens and displays a new plate document. 2 Select FAM from the Dye Layer pop-up menu. 3 Select all wells by clicking and dragging the mouse cursor across the plate document. Note Any data acquired during the reverse transcription reaction is not required for analysis of the TaqMan Cytokine Gene Expression Plate I. However, at least one well must be selected in the setup view to perform a thermal cycling protocol on the 7700 SDS. 4 Select Unknown from the Sample Type pop-up menu. The 7700 SDS software labels all wells as unknown. Reverse Transcription 2-3

18 Performing Reverse Transcription Guidelines Preparing the Reactions Follow the guidelines below to ensure optimal RT performance: Use cdna generated from total RNA samples only. Poly A + RNA samples are not recommended for Cytokine Gene Expression Plate I experiments, because most rrna has been removed from them. Use only random hexamers to reverse transcribe the total RNA samples for use with TaqMan Cytokine Gene Expression Plate I assays. Perform multiple RT reactions in multiple wells if you are using more than 2 µg total RNA. A 100-µL RT reaction will efficiently convert a maximum of 2 µg total RNA to cdna. The following procedure describes the preparation of three different test 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 N ).! CAUTION CHEMICAL HAZARD. TaqMan Reverse Transcription Reagents may cause eye and skin irritation. They 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. 2-4 Reverse Transcription

19 To prepare the reverse transcription reactions: Step Action 1 In a 1.5-mL microcentrifuge tube, prepare a reaction mix for all total RNA samples to be reverse transcribed. Volume (µl) a Per Reaction Final Component Sample Mix (x4) Value RNase-free Water b 10X RT Buffer X 25 mm MgCl mm deoxyntps Mixture µm per dntp Random Hexamers µm RNase Inhibitor U/µL MultiScribe Reverse U/µL Transcriptase (50 U/µL) Total a. If changing the reaction volume, make sure the final proportions are consistent with the recommended values above. b. The volume (µl) of RNase-free water in a 100-µL reaction is RNA sample volume. 2 Label four 1.5-mL microcentrifuge tubes for the three test samples and the calibrator sample. 3 Transfer 60 ng 2 µg (up to µl) of each total RNA sample to the corresponding microcentrifuge tube. 4 If necessary, dilute each total RNA sample to a volume of µl 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 and the calibrator sample. Reverse Transcription 2-5

20 To prepare the reverse transcription reactions: (continued) Step Action 8 Pipet µl of the reaction mix (from step 1) to each MicroAmp Reaction Tube (from step 7). 10X RT Buffer MgCl 2 dntps Mixture Random Hexamers MultiScribe Reverse Transcriptase RNase Inhibitor µl µl µl µl Calibrator Sample 1 Sample 2 Sample 3 9 Transfer µl 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. 2-6 Reverse Transcription

21 Transferring Reverse Transcription Reactions to the Plate. To transfer RT reactions to the plate: Step Action 1 Transfer the contents of the tubes to Individual MicroAmp Optical Tubes or Multiple wells of a MicroAmp Optical 96-Well Reaction Plate as shown below. Calibrator Sample 1 Sample 2 Sample 3 A B C D E F G H GR1309 Note Depending on RNA concentrations and cytokine gene expression levels in your samples, more than one well per sample-specific RT reaction may be necessary to generate enough cdna for the recommended plate configuration (see step 1 on page 3-15). 2 Cap the plate or tubes with MicroAmp Optical Caps. 3 Centrifuge the plate or tubes to force the solution to the bottom and to eliminate any air bubbles from the mixture. Reverse Transcription 2-7

22 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: Step Hexamer Incubation a a. When using random hexamers for first-strand cdna synthesis, the primer incubation step (25 C for 10 min) is necessary to maximize primer-rna template binding. 3 Begin reverse transcription. Reverse Transcription Reverse Transcriptase Inactivation HOLD HOLD HOLD Temp. 25 C 37 C 95 C Time 10min 60min 5min Volume 100 µl IMPORTANT After thermal cycling, store all cdna samples at 15 to 25 C and proceed to Chapter 3, PCR. 2-8 Reverse Transcription

23 PCR 3 Overview 3 About This Chapter This chapter covers PCR, or the amplification of cdna. PCR is the second step in the TaqMan Cytokine Gene Expression Plate I two-step RT-PCR experiment, as described in Cytokine Gene Expression Assay System on page 1-4. In this step, AmpliTaq Gold DNA polymerase amplifies cdna synthesized from the original total RNA sample. Note See Appendix A, Background Information, 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 Creating and Configuring a Cytokine Plate Document 3-2 Setting Thermal Cycling Conditions 3-8 Preparing and Running the PCR Reaction 3-10 PCR 3-1

24 Creating and Configuring a Cytokine Plate Document Cytokine Plate Document Setup Samples (cdna) are arranged on the TaqMan Cytokine Gene Expression Plate I in a format that permits the relative quantification of three samples and a calibrator in duplicate. Each sample is arrayed twice across the plate (i.e., two rows per sample). Figure 3-1 illustrates the sample/assay configuration on the TaqMan Cytokine Gene Expression Plate I. Each well on the cytokine plate contains dried primers and probes for detecting one cytokine cdna target and the endogenous rrna control. The cytokine plate uses two dye layers to detect amplification of both species: a FAM layer and a VIC layer. The FAM dye layer detects the cytokine target sequences, while the VIC dye layer detects the endogenous control. 3-2 PCR

25 A IL-α Calibrator (1) IL-1β Calibrator (2) IL-2 Calibrator (3) IL-4 Calibrator (4) IL-5 Calibrator (5) IL-8 Calibrator (6) IL-10 Calibrator (7) IL-12p35 Calibrator (8) IL-12p40 Calibrator (9) IL-15 Calibrator (10) IFNγ Calibrator (11) TNFα Calibrator (12) B IL-α Calibrator (13) IL-1β Calibrator (14) IL-2 Calibrator (15) IL-4 Calibrator (16) IL-5 Calibrator (17) IL-8 Calibrator (18) IL-10 Calibrator (19) IL-12p35 Calibrator (20) IL-12p40 Calibrator (21) IL-15 Calibrator (22) IFNγ Calibrator (23) TNFα Calibrator (24) C IL-α Sample 1 (25) IL-1β Sample 1 (26) IL-2 Sample 1 (27) IL-4 Sample 1 (28) IL-5 Sample 1 (29) IL-8 Sample 1 (30) IL-10 Sample 1 (31) IL-12p35 Sample 1 (32) IL-12p40 Sample 1 (33) IL-15 Sample 1 (34) IFNγ Sample 1 (35) TNFα Sample 1 (36) D IL-α Sample 1 (37) IL-1β Sample 1 (38) IL-2 Sample 1 (39) IL-4 Sample 1 (40) IL-5 Sample 1 (41) IL-8 Sample 1 (42) IL-10 Sample 1 (43) IL-12p35 Sample 1 (44) IL-12p40 Sample 1 (45) IL-15 Sample 1 (46) IFNγ Sample 1 (47) TNFα Sample 1 (48) E IL-α Sample 2 (49) IL-1β Sample 2 (50) IL-2 Sample 2 (51) IL-4 Sample 2 (52) IL-5 Sample 2 (53) IL-8 Sample 2 (54) IL-10 Sample 2 (55) IL-12p35 Sample 2 (56) IL-12p40 Sample 2 (57) IL-15 Sample 2 (58) IFNγ Sample 2 (59) TNFα Sample 2 (60) F IL-α Sample 2 (61) IL-1β Sample 2 (62) IL-2 Sample 2 (63) IL-4 Sample 2 (64) IL-5 Sample 2 (65) IL-8 Sample 2 (66) IL-10 Sample 2 (67) IL-12p35 Sample 2 (68) IL-12p40 Sample 2 (69) IL-15 Sample 2 (70) IFNγ Sample 2 (71) TNFα Sample 2 (72) G IL-α Sample 3 (73) IL-1β Sample 3 (74) IL-2 Sample 3 (75) IL-4 Sample 3 (76) IL-5 Sample 3 (77) IL-8 Sample 3 (78) IL-10 Sample 3 (79) IL-12p35 Sample 3 (80) IL-12p40 Sample 3 (81) IL-15 Sample 3 (82) IFNγ Sample 3 (83) TNFα Sample 3 (84) H IL-α Sample 3 (85) IL-1β Sample 3 (86) IL-2 Sample 3 (87) IL-4 Sample 3 (88) IL-5 Sample 3 (89) IL-8 Sample 3 (90) IL-10 Sample 3 (91) IL-12p35 Sample 3 (92) IL-12p40 Sample 3 (93) IL-15 Sample 3 (94) IFNγ Sample 3 (95) TNFα Sample 3 (96) Note All wells contain the 18S rrna endogenous control assay in addition to the specified cytokine assays. Figure 3-1 TaqMan Cytokine Gene Expression Plate I: Sample/assay configuration (well numbers shown in parentheses) PCR 3-3

26 Creating a Cytokine Plate Document To create the cytokine plate document using the 7700 SDS software: Step Action 1 Open the 7700 SDS software. The 7700 SDS software opens and displays a new plate document. 2 Create a plate document with the following attributes: Single Reporter 7700 SDS Real Time IMPORTANT The plate document must be configured with the attributes above for the cytokine gene expression assays to work properly. Configuring the FAM Dye Layer To set up the FAM dye layer: Step Action 1 From the Dye Layer pop-up menu, select FAM. 2 Select all wells containing samples by Holding down the mouse button and dragging the pointer diagonally across the plate or Clicking the top-left header box as shown below Click here to select all rows. 3 From the Sample Type pop-up menu, select UNKN - Unknown. The 7700 SDS software labels all selected wells as unknown. 3-4 PCR

27 To set up the FAM dye layer: (continued) Step Action 4 Select rows A and B by Holding down the mouse button and dragging the pointer diagonally across the plate or Holding down the Shift key and clicking the row headers as shown below Hold down the Shift key and click the A and B labels 5 Click in the Sample Name text field, and enter Calibrator. 6 Select rows C and D. 7 Click in the Sample Name text field, and enter your first sample name. 8 Select rows E and F. 9 Click in the Sample Name text field, and enter your second sample name. 10 Select rows G and H. 11 Click in the Sample Name text field, and enter your third sample name. The plate document appears as shown below. PCR 3-5

28 Configuring the VIC Dye Layer The 7700 SDS must be calibrated using the Sequence Detection Systems Spectral Calibration Kit (P/N ) before performing assays with the TaqMan Cytokine Gene Expression Plate I. This spectral calibration kit contains TaqMan VIC and SYBR Green pure dye standards to configure your instrument. If your instrument is not calibrated for the VIC dye, you will be unable to configure the VIC dye layer for the cytokine gene expression assay. To set up the VIC dye layer: Step Action 1 From the Dye Layer pop-up menu, select VIC. 2 From the Sample Type pop-up menu, select Sample Type Setup. The 7700 SDS software displays the Sample Type Setup dialog box. 3 From the Internal Positive (IPC+) Reporter pop-up menu, select VIC. The 7700 SDS software displays VIC in the Reporter box for the Internal Positive Control entry as shown below. IMPORTANT The TaqMan VIC dye must be configured as a pure dye for it to appear on the Reporter pull-down menu. See the ABI PRISM 7700 Sequence Detection System User Bulletin #4: Generating New Spectra Components (P/N ) to configure TaqMan VIC as a pure dye. 3-6 PCR

29 To set up the VIC dye layer: (continued) Step Action 4 Click OK. The SDS software returns to the Setup Plate view. 5 Select all wells containing samples. 6 From the Sample Type pop-up menu, select IPC+ (Internal Positive Control). 7 Follow step 4 through step 11 in Configuring the FAM Dye Layer, which begins on page 3-4. The plate document appears as shown below. PCR 3-7

30 Setting Thermal Cycling Conditions Setting the Software To configure the thermal cycling conditions for your instrument: Step Action 1 Click Thermal Cycler Conditions from the plate document. The software displays the Thermal Cycler Conditions dialog box. 2 Click individual Time and Temperature text fields and enter the following settings as shown in the illustration below: Amperase UNG Incubation PCR Cycles Each of 40 Cycles Denature Anneal/ Extend AmpliTaq Gold Activation Stage HOLD HOLD CYCLE Temperature 50 C 95 C 95 C 60 C Time 2 min 10 min 15 sec 1 min Volume 50 µl IMPORTANT The 2-min, 50 C step is required for optimal AmpErase UNG enzyme activity. The 10-min, 95 C step is required to activate AmpliTaq Gold DNA Polymerase. See the ABI PRISM 7700 Sequence Detection System User s Manual (P/N ) for more information on programming the thermal cycler conditions. 3-8 PCR

31 To configure the thermal cycling conditions for your instrument: Step Action 3 Click on the Sample Volume text field and enter 50 µl. 4 Click Show Data Collection. The 7700 SDS software displays the Data Collection view. 5 Place Data Collection icons at all stages of the run by clicking on the plateaus for each stage. See the user s manual for more information on adding and deleting Data Collection icons to the thermal cycler conditions. 6 Click OK. 7 Click Show Analysis. The 7700 SDS software displays the Analysis Plate view. PCR 3-9

32 Preparing and Running the PCR Reaction Guidelines Preparing the PCR Reaction Mix For optimal PCR performance follow these guidelines: Calibrate the 7700 Sequence Detection System with the Sequence Detection System Calibration Kit (P/N ) before performing assays. Do not remove the TaqMan Cytokine Gene Expression Plate I from its foil packaging until you are ready to load the PCR reaction mix and cdna. Excessive exposure to light can result in probe degradation. Prepare the PCR reaction mixture for each sample in a separate microcentrifuge tube before adding the cdna and aliquoting into the TaqMan Cytokine Gene Expression Plate I. The volume of PCR reaction mix and cdna must be 50 µl per well. Volumes other than 50 µl will result in incorrect primer and probe concentrations. Load 10 ng 1µg of total RNA (converted to cdna) per well to utilize the full dynamic range of the plate. Carefully preparing the PCR reaction mix ensures that you will obtain accurate relative quantification values.! CAUTION 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. To prepare the PCR reaction mix: Step Action 1 Label a 1.5-mL microcentrifuge tube for each sample and for the calibrator PCR

33 To prepare the PCR reaction mix: (continued) Step Action 2 Review the following table to determine the volume of RNase-free water that you will be using in each PCR reaction. Component Volume/Well (µl) Final Concentration RNase-free water 25 y TaqMan Universal PCR 25 1X Master Mix (2X) Primers and probe Endogenous Reference Preloaded 1X Primers and probe Target Preloaded 1X Cytokine cdna template y Total volume 50 1X Note We recommend using 20 µl of RNase-free water and 5 µl of cdna in each well. PCR 3-11

34 To prepare the PCR reaction mix: (continued) Step Action 3 Pipette the TaqMan Universal PCR Master Mix (2X) and RNase-free H 2 0 into the microcentrifuge tubes for the calibrator and for each sample. See the table below for volumes. Reagent Volume (µl) for 1 Well Volume (µl) for 26 Wells a TaqMan Universal PCR Master Mix RNase-free H b 520 b Total volume a. Total includes additional volume for two extra wells to provide a margin for experimental error. b. RNase-free H 2 O volumes adjusted to accommodate 5-µL cdna samples. Master Mix (2X) RNase-free H 2 O GR1278b Calibrator Sample 1 Sample 2 Sample PCR

35 Adding the cdna to PCR Reaction Mix In this step you transfer the cdna samples produced in the RT step to the microcentrifuge tube containing the PCR reaction mix. To add the cdna to the sample and PCR reaction mix: Step Action 1 Obtain the MicroAmp 96-Well Reaction Plate containing the cdna samples produced in the RT step. 2 IMPORTANT Slowly and carefully remove the caps from the MicroAmp 96-Well Reaction Plate to avoid contamination of the reverse transcription products. PCR 3-13

36 To add the cdna to the sample and PCR reaction mix: (continued) Step Action 3 Transfer the cdna samples to the appropriate microcentrifuge tubes containing the PCR reaction mix. Use the proportions shown in the table below. Reagent TaqMan Universal PCR Master Mix and RNase-free H 2 0 mixture Volume (µl) for 1 Well Volume (µl) for 26 Wells a cdna Total volume a. Total includes additional volume for two extra wells to provide a margin of error for the experiment. A MicroAmp 96-well reaction plate B cdna from RT Step C D E F G H GR1302 Calibrator Sample 1 Sample 2 Sample 3 4 Cap the microcentrifuge tubes and mix the solution by gentle inversion. 5 Centrifuge the tubes to force all reagents to the tube bottoms and eliminate any air bubbles in the mixture PCR

37 Loading the Cytokine Gene Expression Plate I To load the PCR reaction mix with cdna into the Cytokine Gene Expression Plate: Step Action 1 Using a positive displacement pipette, transfer 50 µl of the PCR reaction mix with cdna into each appropriate well of the Cytokine Gene Expression Plate I. cdna/master Mix/ RNase-free H 2 O Mixture Sample 3 Sample 2 Sample 1 Calibrator A B C D E F G H Cytokine Gene Expression Plate I GR Seal the wells with MicroAmp Optical Caps or a MicroAmp Optical Adhesive Cover. 3 Centrifuge the 96-well plate to force all reagents to the bottom of the wells and eliminate any air bubbles in the mixture. 4 Transfer the plate to the sequence detector sample block. Note If you have not yet programmed the thermal cycling conditions, see Setting Thermal Cycling Conditions on page Slide the sample block cover over the sample block and tighten the lid. 6 From the Setup view, click the Show Analysis button to toggle to the Analysis view. 7 Click Run to begin thermal cycling. PCR 3-15

38 3-16 PCR

39 Data Analysis 4 Overview 4 About This Chapter This chapter covers data analysis. Before calculating relative quantification values from the results of the TaqMan Cytokine Gene Expression Plate I, you must analyze the raw data and export it to a results file. The analysis procedure consists of setting threshold values for the FAM and VIC dye layers and eliminating outlying amplification. IMPORTANT The sequence of events listed below is crucial to data analysis and will produce errors if performed out of order. If you set the threshold value before setting the baseline value, the resulting C T values may be invalid and produce errors when calculating relative quantification values. Setting the baseline for the FAM dye layer Setting the threshold for the FAM dye layer Setting the baseline for the VIC dye layer Setting the threshold for the VIC dye layer In This Chapter The following topics are discussed in this chapter: Topic See Page Before the Analysis 4-2 Setting the Baseline Values 4-3 Setting the Baseline for the FAM Dye Layer 4-5 Setting the Threshold Value for the FAM Dye Layer 4-7 Setting the Baseline for the VIC Dye Layer 4-11 Setting the Threshold Value for the VIC Dye Layer 4-11 Data Analysis 4-1

40 Before the Analysis Activating Spectral Compensation For all real-time runs, set the spectral compensation to On during analysis of multiplex PCR assays. Activating spectral compensation provides improved spectral resolution for multi-reporter applications. To activate spectral compensation: Step Action 1 Select Instrument > Diagnostics > Advanced Options. The Advanced Options dialog box opens. 2 Click the Use Spectral Compensation for Real Time checkbox from the Miscellaneous box. Click here 3 Click OK. 4-2 Data Analysis

41 Setting the Baseline Values Displaying Results on an Amplification Plot To display the results on an amplification plot for data analysis: Step Action 1 Select Analysis > Analyze. The 7700 SDS software analyzes the raw data and displays an amplification plot. 2 If the 7700 SDS software doesn t display the amplification plot, select Analysis > Amplification Plot. The 7700 SDS software displays an amplification plot (log R n vs. Cycle), as shown below. Data Analysis 4-3

42 Baseline Basics The baseline is a range of cycles that you must define before the 7700 SDS software detects the amplification of PCR product. For standard PCR assays, the 7700 SDS software uses a default range of cycles 3 15 to establish the baseline for a PCR assay. Figure 4-1 illustrates the important characteristics of the baseline. Initial amplification of cdna (baseline ends before this point) Default baseline (cycles 3 15) Product amplification Figure 4-1 Example of a linear-scale amplification plot Because of the abundance of rrna, low C T values are obtained in TaqMan RT-PCR applications with this target. During data preparation, if the amplification plot appears differently than as shown in Figure 4-1, then it is necessary to change the baseline numbers. Guidelines for Setting the Baseline Correct placement of the baseline is a crucial step for data analysis. Follow the guidelines below to ensure the baseline is set properly. Set the baseline such that the amplification curve growth begins at a cycle number that is greater than the highest baseline number. Do not adjust the default baseline if the amplification curve growth begins after cycle 15. For example, in Figure 4-1 the default baseline can be used because the initial amplification value occurs well after cycle Data Analysis

43 Setting the Baseline for the FAM Dye Layer Viewing the FAM Baseline To view the baseline for the FAM dye layer: Step Action 1 From the Reporter pop-up menu, select FAM. 2 Double-click the Rn label on the Y-axis of the amplification plot. The Scale dialog box appears. 3 Click the Linear Scale radio button to graph the data on a linear scale. 4 Click OK. The amplification plot appears in a linear scale format. Data Analysis 4-5

44 Setting the FAM Baseline To set the baseline for the FAM dye layer: Step Action 1 Identify the components of the linear scale amplification plot, as illustrated in Figure 4-1 on page Click the Stop text field in the Baseline box. 3 Follow the guidelines and choose from one of the following actions: 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. 4 Click Update Calculations. The 7700 SDS software updates the C T and Standard Deviation values. 4-6 Data Analysis

45 Setting the Threshold Value for the FAM Dye Layer Threshold Value Basics The default threshold value is the average standard deviation of R n within the defined baseline region, multiplied by an adjustable factor. The 7700 SDS software calculates the threshold value as 10 standard deviations from the baseline. For that reason, the baseline must be set before you adjust the threshold value. Figure 4-2 illustrates the important characteristics of the threshold. Exponential amplification phase Plateaued region Threshold value Background (spectral noise) Product amplification curves Figure 4-2 Example of a logarithmic scale amplification plot The threshold value must be set within the exponential phase of the logarithmic scale amplification plot. The exponential phase occurs within the range of datapoints that increase linearly when graphed on the plot. Data Analysis 4-7

46 Viewing the FAM Threshold Value To view the threshold value for the FAM dye layer: Step Action 1 Double-click the Rn label on the Y-axis of the graph. The Scale dialog box appears. 2 Click the Logarithmic Scale radio button to graph the data on a logarithmic scale. 4-8 Data Analysis

47 To view the threshold value for the FAM dye layer: (continued) Step Action 3 Click OK. The amplification plot appears in logarithmic format. Data Analysis 4-9

48 Setting the FAM Threshold Value To set the threshold value for the FAM dye layer: Step Action 1 Identify the components of your amplification curve as illustrated in Figure 4-2 on page 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 Threshold cursor Set threshold within this range Background Note You can reset the threshold to the default value at any time by clicking Suggest in the Threshold box. 3 Click Update Calculations. The 7700 SDS software updates the C T and Standard Deviation values Data Analysis

49 Setting the Baseline for the VIC Dye Layer Setting the VIC Baseline To set the baseline for the endogenous control assay, follow the procedure for Setting the Baseline for the FAM Dye Layer on page 4-5, with the following change: Step Action 1 From the Amplification Plot dialog box, select VIC from the Reporter pop-up menu. The 7700 SDS software displays the VIC Endogenous Control Assay Amplification Plot. IMPORTANT Baseline and threshold values are valid only within a specific dye layer. Therefore, baseline and threshold values set within the FAM dye layer will not be changed when modifying baseline and threshold values within the VIC dye layer. This enables accurate and independent baseline and threshold values to be set for the FAM and VIC dye layers. Setting the Threshold Value for the VIC Dye Layer Setting the VIC Threshold Value To set the threshold value for the endogenous control assay, follow the procedure for Setting the Threshold Value for the FAM Dye Layer on page 4-7. Data Analysis 4-11

50

51 Calculating Relative Quantification 5 5 Overview About This Chapter This chapter covers the calculation of relative quantification values. The relative quantification values for the cytokine gene expression assays can be calculated when accurate C T values have been obtained from both dye layers in the assay. You must calculate the values using a spreadsheet application. Applied Biosystems recommends using a professional spreadsheet software package such as Microsoft Excel to analyze the results from the TaqMan Cytokine Gene Expression Plate I. Although calculation of relative quantification values can be done manually, spreadsheet packages speed the process considerably. This section explains how to calculate relative quantification values from C T values with and without the use of a spreadsheet application. Both procedures provide instructions for calculating relative quantification values and include example calculations in Microsoft Excel. In This Chapter The following topics are discussed in this chapter: Topic See Page Exporting and Viewing the Results File 5-2 Analyzing Multiplex PCR Results (Comparative C T Method) 5-5 Manually Calculating Relative Quantification Values 5-6 Calculating the Relative Quantification Using a Spreadsheet 5-8 Interpreting Results 5-22 Calculating Relative Quantification 5-1

52 Exporting and Viewing the Results File Creating a Results File To analyze data from TaqMan Cytokine Gene Expression Plate I using the Comparative C T Method, the results must be exported into a data file. The 7700 SDS software can export raw data in a tab-delimited format. Figure 5-1 shows the format of a typical 7700 SDS exported results data file. Note Data from the FAM dye layer appears in rows Data from the VIC dye layer follows in rows Well number Reporter dye Threshold cycle value Figure SDS exported results data file 5-2 Calculating Relative Quantification

53 Exporting Data to a Results File To export the data from the cytokine gene expression assay: Step Action 1 Select File > Export > Results. 2 Click the Export result data as text box, and enter a name for the exported file. 3 Click the Export All Wells radio button to save the data from all wells to the data file. 4 Click Export when you are done. The 7700 SDS software exports the data to a Microsoft Excel spreadsheet document. The data file icon is shown below. 5 Close the 7700 SDS software. Calculating Relative Quantification 5-3

54 Viewing the Results File The 7700 SDS software exports the data from a PCR run in a tab-delimited format. To view the exported results file: Step Action 1 Open the spreadsheet software. 2 Select File > Open. 3 Select the exported results file, and click Open. The cytokine plate results file spreadsheet appears, as seen in Figure 5-1 on page Calculating Relative Quantification

55 Analyzing Multiplex PCR Results (Comparative C T Method) Basics of the Relative Quantification Calculation Relative quantification of cytokine gene expression is calculated from the threshold cycle (C T ) values for each well on the cytokine plate. The threshold cycle is the cycle at which a statistically significant increase in R n is first detected. Thus, wells with higher initial template concentrations reach the threshold value at lower cycle numbers during PCR than wells containing lower initial template concentrations. Because amplicons designed and optimized according to Applied Biosystems guidelines have equivalent efficiencies approaching 100 percent, each cycle in the PCR reaction corresponds to a twofold increase in PCR product. Therefore a change in threshold cycle number of 1 equates to a twofold difference in initial template concentration. For example, if the C T increases by one, the initial template concentration decreases twofold. Conversely, if the C T decreases by one, the initial template concentration doubles. This property is used in the calculation of relative quantification values for the cytokine and internal control target sequences in each well. Calculating Relative Quantification 5-5

56 Manually Calculating Relative Quantification Values Calculating Relative Quantification Manually Relative gene expression levels can be calculated manually from the exported results file. The following procedure describes the calculation of relative quantification values of TNFα gene expression for a single sample and its calibrator. Refer to Figure 3-1 on page 3-3 for sample and calibrator positions on the cytokine plate. IMPORTANT Do not use wells with FAM C T values greater than or equal to 36 or VIC C T values greater than or equal to 23. They may produce inaccurate and unreliable relative quantification values. Note If VIC C T values are consistently equal to or greater than 23 for any sample, Applied Biosystems recommends that you increase the amount of initial total RNA (converted to cdna) used in each reaction. To manually calculate the relative cytokine gene expression levels: Step Action Example 1 Subtract the VIC C T values from the FAM C T values to calculate C T for the calibrator and samples in each cytokine gene expression assay. C T = C T (FAM) C T (VIC) Subtract the VIC C T value from the FAM C T value for each well in the TNFα cytokine column. Calibrator Unknown Well C T (FAM) C T (VIC) C T Average the C T values for duplicate wells of the calibrator sample for each cytokine column. Average C T (calibrator) = C T (calibrator A) + C T (calibrator B) 2 Average the calibrator wells from step 1. C T (Well 12) + C T (Well 24) = Average C T (Calibrator) 2 (7.67) + (7.6) = Calculating Relative Quantification

57 To manually calculate the relative cytokine gene expression levels: (continued) Step Action Example 3 Subtract the Average C T(calibrator) values from the C T values of samples in its column to calculate their C T (sample) values. C T (sample) = C T (sample) Average C T (calibrator) Subtract the calibrator Average C T value (step 2) from the sample C T values (step 1). Unknown Calibrator Well (averaged) C T(sample) Avg C T(calibrator) C T Average the C T values for duplicate wells of each cytokine sample. Average C T (sample) = Average the unknown wells from step 3. C T (Well 84) + C T (Well 96) = average C T (sample) 2 C T (sample) + C T (sample) This operation normalizes the number of target mrna molecules to the number of rrna molecules. 5 Enter the Average C T values for each sample into the equation below to calculate the relative quantification values. Relative quantification = 2 Average C T 2 Note See page 11 of the ABI PRISM 7700 Sequence Detection System User Bulletin #2: Relative Quantification of Gene Expression for the derivation of the relative quantification equation = Enter the Average C T values from step 4 into the relative quantification equation shown below. 2 Average C T = relative quantification 2 Average C T (sample) = 2 ( 0.775) = Average C (calibrator) T = 2 ( 0.0) = 1 Therefore, the TNFα cytokine mrna concentration in the sample well is nearly twice that of the control. Note Because the values for the calibrator wells are always subtracted from themselves, their relative quantification values are always 1. Calculating Relative Quantification 5-7

58 Calculating the Relative Quantification Using a Spreadsheet Using Microsoft Excel to Calculate Relative Quantification The following procedure demonstrates how to analyze exported data from a cytokine gene expression assay using the Comparative C T Method and Microsoft Excel software. Other software packages can be used to calculate relative quantification values, although the keystrokes and command pathways in the procedure may differ. This procedure is valid only when the cytokine plate is configured as shown in Figure 3-1 on page 3-3 (i.e., using three samples and a calibrator). Any deviation from this recommended configuration will require you to modify the following procedure. IMPORTANT Applied Biosystems support personnel are not responsible for supporting plate configurations other than the one described in this protocol. For that reason, Applied Biosystems strongly recommends you adhere to the configuration shown in Figure 3-1 on page 3-3. Transferring Data from the Results File To transfer data from the results file to the spreadsheet: Step Action 1 In Microsoft Excel select File > New. A new spreadsheet appears. 2 From the Window menu, select the cytokine plate results file. The cytokine plate results spreadsheet reappears. 5-8 Calculating Relative Quantification

59 To transfer data from the results file to the spreadsheet: (continued) Step Action 3 Select the cells that contain the well number data (A1 through A97) by clicking and dragging the mouse cursor down the spreadsheet document. 4 Select Edit > Copy. 5 From the Window menu, select the new spreadsheet. The new spreadsheet file reappears. 6 Click in cell A1. 7 Select Edit > Paste. Excel pastes the data into the new spreadsheet. Calculating Relative Quantification 5-9

60 To transfer data from the results file to the spreadsheet: (continued) Step Action 8 Repeat steps 3 7 to copy the FAM C T values in cells F1 through F97 from the cytokine plate results file, and paste them into cells B1 B97 of the new Excel file. 9 Double-click the B1 cell; enter C T FAM and press Return. 10 Repeat steps 3 7 to copy the VIC C T values in cells F111 through F207 from the cytokine plate results file, and paste them into cell C1 of the new Excel file. 11 Double-click the C1 cell and enter C T VIC and press Return Calculating Relative Quantification

61 Calculating C T C T is calculated using the equation: C T = C T (FAM) C T (VIC). To calculate C T using the spreadsheet, the formula must be entered into column D. To enter the equation into the spreadsheet: Step Action 1 Click cell D2. 2 Press the = key. 3 Enter the equation B2-C2 as shown below and press Return. 4 Click cell D2. 5 Select Edit > Copy. 6 Select cells D3 through D97 by clicking and dragging the mouse cursor down the spreadsheet document. 7 Select Edit > Paste. Excel pastes copies of the equation into cells D2 D97 of the new spreadsheet. The program automatically calculates C T values for all of the wells. Calculating Relative Quantification 5-11

62 To enter the equation into the spreadsheet: (continued) Step Action 8 Double-click the D1 cell and enter C T. 9 Select File > Save as. Save the spreadsheet document. Deleting Invalid Wells For any assay, some samples may not amplify sufficiently and some may fail to amplify at all. The large C T values from these wells are not suitable for generating accurate relative quantification values because they are outside the proven linear dynamic range of the assays. Thus, the data from these wells must be eliminated from the calculations. IMPORTANT Do not use data from wells with FAM C T values greater than or equal to 36 or VIC C T values greater than or equal to 23. They may produce inaccurate and unreliable relative quantification values. To eliminate the invalid rows: Step Action 1 Select rows containing FAM C T values greater than or equal to 36, as shown below. Clear all wells with FAM C T 36 or VIC C T Calculating Relative Quantification

63 To eliminate the invalid rows: (continued) Step Action 2 Select Edit > Clear > All. The spreadsheet deletes the data from the selected cells. 3 Repeat steps 1 2 for all rows containing VIC C T values greater than or equal to 23. Creating a C T Table Now that C T values have been calculated from the cytokine plate data, the results must be organized under the cytokines they detect. This step is necessary to organize the results in a tabular format. To create a C T table: Step Action 1 Click on cell F2 and enter Ct Cytokine Plate Results. 2 Enter the letters A H into cells G4 N4 of the spreadsheet as shown below. Calculating Relative Quantification 5-13

64 To create a C T table: (continued) Step Action 3 Enter the names of the cytokines into cells F5 F16 as shown below. 4 Enter the following equations into the spreadsheet: Cell Equation G5 =D2 H5 =D14 I5 =D26 J5 =D38 K5 =D50 L5 =D62 M5 =D74 N5 =D86 5 Select and copy cells G5 N5. 6 Select cells G6 N16 by clicking and dragging the mouse pointer across the spreadsheet. 7 Paste the copied cells to cells G6 N16 of the table Calculating Relative Quantification

65 Deleting Invalid Wells from the C T Table Before creating a C T table, you must clear all wells with C T values of 0 from the C T table. These values are a result of the rows cleared in the procedure on page 5-12, step 1. In the following sections, the C T values from these wells will be averaged with the values from their duplicate wells to calculate relative quantification values for the samples. If these wells are not cleared from the table, their C T values could falsely decrease the real sample C T values. These errors will continue through into the relative quantification calculation. To eliminate the cells with C T values of 0: Step Action 1 Hold down the Command key, and click on all cells with C T values of 0. 2 Select Edit > Clear > All. 3 Select and clear all rows with blank calibrator cells in both the A and B columns. Relative quantification values cannot be calculated without using a calibrator as a basis for comparison. Therefore, if both calibrator wells are blank, the corresponding row is invalid and must be cleared. This row must be cleared because both calibrator wells failed to amplify Calculating Relative Quantification 5-15

66 Calculating C T from C T To calculate the C T values for the samples, the C T of the calibrator wells must be averaged and subtracted from the corresponding cytokine targets in each column. To calculate C T : Step Action 1 Click on cell F18 and enter Ct Cytokine Plate Results ( Ct [sample] Ct[calibrator]). 2 Enter the following headings into cells G20 M20: Cell G20 H20 I20 J20 K20 L20 M20 Text Calibrator a C D E F G H a. Equal to the average C T of the calibrator replicate wells. The table should resemble the sample below. 3 Copy and paste the cytokine labels from the C T table to cells F21 F Calculating Relative Quantification

67 To calculate C T : (continued) Step Action 4 Enter the following equations into cells G21 M21: Cell G21 H21 I21 J21 K21 L21 M21 Equation =AVERAGE(G5:H5) =I5-G21 =J5-G21 =K5-G21 =L5-G21 =M5-G21 =N5-G21 5 Select and copy cells G21 M21. 6 Paste the copied cells to cells G22 M32 of the C T table. The software updates the spreadsheet automatically. Note The spreadsheet software will display some entries as #DIV/0! These are a consequence of the invalid wells. Calculating Relative Quantification 5-17

68 Averaging To average the sample C T values and calculate C T(Calibrator) : Sample C T Step Action 1 Click in cell F34 and enter Ct Cytokine Plate Results Averages. 2 Enter the following headings into cells G36 J36: Cell Text G36 Calibrator H36 Sample 1 I36 Sample 2 J36 Sample 3 The table should resemble the sample below. 3 Copy and paste the cytokine labels from the C T table to cells F37 F48. 4 Enter the following equations into cells G37 J37 of the spreadsheet: Cell G37 H37 I37 J37 Equation =G21-G21 =AVERAGE(H21:I21) =AVERAGE(J21:K21) =AVERAGE(L21:M21) 5-18 Calculating Relative Quantification

69 To average the sample C T values and calculate C T(Calibrator) : Step Action 5 Select and copy cells G37 J37. 6 Paste the copied cells to cells G38 J48 of the Average C T table. The software automatically calculates Average C T values. Calculating Relative Quantification from C T The Comparative C T method uses arithmetic formulas to permit the relative quantification of gene expression for multiple targets in multiple samples with TaqMan assays. The amount of target, normalized to an endogenous reference and relative to a calibrator, is given by Relative quantification = 2 Average C T which describes the amount of normalized target in the test sample relative to the amount of normalized target in the calibrator sample. To determine the relative quantification values: Step Action 1 Click in cell F50 and enter Relative Quantification Results. Calculating Relative Quantification 5-19

70 To determine the relative quantification values: (continued) Step Action 2 Enter the following headings into cells G52 J52: Cell Text G52 Calibrator H52 Sample 1 I52 Sample 2 J52 Sample 3 The table should resemble the sample below. 3 Copy and paste the cytokine labels from the C T table to cells F53 F Calculating Relative Quantification

71 To determine the relative quantification values: (continued) Step Action 4 Enter the following equations into cells G53 J53 of the spreadsheet: Cell G53 H53 I53 J53 Equation =2^(-G37) =2^(-H37) =2^(-I37) =2^(-J37) Note For the calibrator sample, the relative quantification value is always 1. Relative quantification of gene expression is therefore expressed as an n-fold increase or decrease in expression relative to the calibrator. 5 Select and copy cells G53 J53. 6 Paste the copied cells to cells G54 J64 of the table. The software automatically calculates the relative quantification values for the calibrator and samples. Calculating Relative Quantification 5-21

72 Interpreting Results Graphing the Data The results of the TaqMan Cytokine Gene Expression Plate I assays are expressed as the normalized messenger RNA level in a test sample relative to the normalized level of that message in the calibrator sample. To best analyze these results, Applied Biosystems suggests graphing the results from the data analysis. To graph the results of your calculations: Step Action 1 Select cells F52 J64 as shown in the following illustration. 2 Select Insert > Chart > 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. IMPORTANT View the graph on a logarithmic scale Calculating Relative Quantification

73 Interpreting the Graph When graphed, your results should appear as shown in the figure below. The relative quantification values of each cytokine gene expression assay are expressed in orders of magnitude greater or less than the calibrator. Note Data is not displayed for cytokines IL-1β, IL-12p35, and IL-12p40 due to the removal of data from their invalid calibrator wells (see step 1 of the procedure on page 5-12). Demonstrating Performance with TaqMan Control Total RNA TaqMan Control Total RNA (Human) is available for demonstrating the performance of the TaqMan Cytokine Gene Expression Plate I. Figure 5-2 displays the typical cytokine gene expression profile for this sample. Calculating Relative Quantification 5-23