Applied Biosystems TaqMan Array Microfluidic Cards

Transcription

1 Performing Rapid Cycling Gene Quantitation on TaqMan Array Microfluidic Cards Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin JULY 2010 SUBJECT: In this user bulletin Performing Rapid Cycling Gene Quantitation on TaqMan Array Microfluidic Cards This user bulletin describes a rapid cycling protocol to perform gene quantitation with TaqMan Array Microfluidic cards. Overview Protocol for rapid cycling with TaqMan Array Microfluidic Cards Create and set up a new plate document Prepare the TaqMan Array Run the TaqMan Array Analyze the experiment Review the data Perform downstream analysis Related information System requirements Instrument setup Materials not provided Overview This assay protocol describes the steps to perform gene quantitation for TaqMan Gene Expression Assays and TaqMan MicroRNA Assays with a rapid cycling protocol, using a standard master mix on Applied Biosystems TaqMan Array Microfluidic Cards. It describes the steps for launching the SDS software and setting up a new plate document, then preparing the TaqMan Arrays, then analyzing the results. The section Create and set up a new plate document describes changes to make in the thermal cycling program that reduce the cycling time from two hours to approximately one hour, thus doubling the throughput of the arrays. Analysis has demonstrated that the number of cycles can be reduced without affecting the relative expression levels. IMPORTANT! The performance of Applied Biosystems TaqMan Gene Expression Assays and TaqMan MicroRNA Assays on Custom TaqMan Arrays and TaqMan Array Gene Signature Panels has been verified using rapid cycling conditions and with both: TaqMan Universal Master Mix II, no UNG TaqMan Universal PCR Master Mix, no AmpErase UNG We do not recommend using a TaqMan Fast master mix with the rapid cycling protocol. The performance of rapid cycling on TaqMan Arrays has been verified for quantitative applications only and not for endpoint applications, such as allelic discrimination. Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin 1

2 Protocol for rapid cycling with TaqMan Array Microfluidic Cards Protocol for rapid cycling with TaqMan Array Microfluidic Cards Create and set up a new plate document 1. Launch SDS v2.1 or later by double-clicking (SDS) on the computer desktop. 2. Create a plate document for TaqMan Array rapid cycling gene quantitation: a. In the SDS software, click (or select File New). Alternatively, to create a new plate document using the New Plate Wizard, follow the instructions in the Applied Biosystems 7900HT Fast Real-Time PCR System Relative Quantitation Using Comparative C T Getting Started Guide (PN ). b. In the New Document dialog box, select the settings shown below, then click OK. 3. Click the Instrument tab, then click the Thermal Profile tab. 2 Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin

3 Performing Rapid Cycling Gene Quantitation on TaqMan Array Microfluidic Cards Protocol for rapid cycling with TaqMan Array Microfluidic Cards 4. Modify the default thermal profile settings: 4a: Delete Stage 1 4b: Change to 37 repeats 4c: Change to 00:05 seconds 4d: For mrna assay, change to 00:30 seconds. For MicroRNA assay, change to 00:45 seconds. Note: Applied Biosystems validation studies have shown that reducing the denaturation and annealing/extension times has no effect on relative expression levels compared to the standard thermal cycling times on TaqMan Arrays. a. Delete Stage 1 (50 C for 2 minutes) by clicking into (or near) the field boxes under that stage. The line turns red to show that the stage is selected for editing. Then click Delete Step. Stage 2 (94.5 C for 10:00 minutes) becomes Stage 1. Note: If you choose to use TaqMan Universal PCR Master Mix with AmpErase UNG, do not delete this step. Note that we have not validated rapid cycling with master mix that contains UNG. b. Reduce the number of repeats from 40 to 37. (See Overview on page 1 for explanation.) c. Reduce the time for the first step in Stage 2 (denaturation at 97 C) from 00:30 seconds to 00:05 seconds. Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin 3

4 Protocol for rapid cycling with TaqMan Array Microfluidic Cards d. Reduce the time for the second step in Stage 2 (extension at 59.7 C) from 01:00 minutes: mrna assay reduce to 00:30 seconds MicroRNA assay reduce to 00:45 seconds mrna assay MicroRNA assay 5. Click the Ramp Rate tab. Change the ramp rate for the first step (97 C denaturation) in Stage 2 from 50% to 100%. 6. Import the SDS Setup File (.txt) from the Array Information CD that ships with the TaqMan Array. 4 Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin

5 Performing Rapid Cycling Gene Quantitation on TaqMan Array Microfluidic Cards Protocol for rapid cycling with TaqMan Array Microfluidic Cards For more information on setting up the Plate Document, see the TaqMan Array Micro Fluidic Cards User Guide (PN ). IMPORTANT! Do not modify the contents of the SDS Setup File. Modification can corrupt the file, making it unusable. The SDS setup file contains information specific to your TaqMan Array. The SDS software uses the information in the setup file to automatically configure each assay detector and place it in the correct well. 7. Save the plate document: a. Click (or select File Save As). b. For Files of Type, select: SDS 7900HT Document (*.sds) to save the plate document as a file SDS 7900HT Template Document (*.sdt) to save the plate document as a template We recommend saving the plate document as a template if you want to create plate documents for a series of TaqMan Array cards with identical assay configurations and run under rapid cycling conditions. c. Locate the folder where you want to save the plate document file. d. In the File Name field, enter a name for the plate document. e. Click Save. Prepare the TaqMan Array 1. Prepare the PCR reaction mix: For a more detailed description, refer to the TaqMan Array Micro Fluidic Cards User Guide (PN ) or watch the video Demonstration of the TaqMan Low Density Array at taqman_tlda/tlda_1.cfm. a. For each sample, determine the total number of reservoirs to be filled, based on the format of your TaqMan Array card. b. Use the table below to calculate the total volumes required for each reaction component. Component cdna sample TaqMan Universal Master Mix II, No UNG (2 ) or TaqMan Universal PCR Master Mix (No AmpErase UNG) (2 ) Volume (μl) per Fill Reservoir Total Volume 100 For recommended amount of cdna, see specific protocols: Megaplex Pools for microrna Expression Analysis Protocol (PN ) for TaqMan microrna assays; TaqMan Array Micro Fluidic Cards User Guide (PN ) for TaqMan Gene Expression assays. You can preamplify your sample using TaqMan PreAmp Master Mix (PN ) when the sample may be limiting or has low-expressing targets. Use only one type of Master Mix per study Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin 5

6 Protocol for rapid cycling with TaqMan Array Microfluidic Cards c. If the cdna samples are frozen, thaw them on ice. Resuspend the cdna samples by inverting the tube, then gently vortexing. d. Mix the master mix thoroughly by swirling the bottle. e. For each sample, label a 1.5-mL microcentrifuge tube, then add the required volume of each component to the labeled tube. f. Cap the microcentrifuge tubes, then gently vortex the tubes to thoroughly mix the solution. g. Briefly centrifuge the tubes to spin down the contents and eliminate air bubbles. 2. Fill the TaqMan Array Card: a. Allow the TaqMan Array card to reach room temperature, then carefully remove it from its packaging. b. Place the TaqMan Array card on a lab bench, with the foil side down. c. Load 100 μl of the desired sample-specific PCR reaction mix into a 100-μL micropipette. d. Hold the micropipette in an angled position and place the tip in the fill port. e. Dispense the sample-specific PCR reaction mix so that it sweeps in and around the fill reservoir toward the vent port. 3. Centrifuge the card (Refer to the TaqMan Array Micro Fluidic Cards User Guide (PN ) for more information.): a. Put an empty custom centrifuge bucket on a lab bench, with the label facing you. b. Insert TaqMan Array cards into the card holder, making sure that the fill reservoirs project upward out of the card holder. c. Put a filled card holder into the bucket so that the This Side Out label faces the front of the bucket. d. Put the buckets into the centrifuge. e. Run the centrifuge. f. Remove the TaqMan Array cards. g. Examine the TaqMan Array cards to be sure that the filling is complete. The amount of PCR reaction mix remaining in the fill reservoirs should be consistent from reservoir to reservoir. 4. Seal the TaqMan Array card: a. Position the sealer. b. Insert a TaqMan Array card into the sealer. c. Push the carriage across the base of the sealer in the direction of the arrows. d. Remove the sealed TaqMan Array Card from the sealer. e. Inspect the TaqMan Array Card for proper sealing. f. Using scissors, trim the fill strip from the TaqMan Array Card. 6 Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin

7 Performing Rapid Cycling Gene Quantitation on TaqMan Array Microfluidic Cards Protocol for rapid cycling with TaqMan Array Microfluidic Cards Run the TaqMan Array 1. Start the SDS Software (v2.1 or later). 2. Click (or File Open). In the dropdown menu, select the file that you created in Create and set up a new plate document. IMPORTANT! Make sure that the array is set up correctly. The SDS setup file must correspond to the assays in the wells, while each plate document setup must correspond exactly to the layout of samples in the wells of the array card. 3. Perform the run: a. In the open SDS file, select the Instrument tab. IMPORTANT! Select the Thermal profile tab and make sure that the cycling conditions are the rapid-cycling parameters that you set in step 3 on page 2 through step 5 on page 4. b. Verify that the instrument tray is inside the instrument. If you are running: One array card Proceed to step c. More than one array card In the SDS Automation Controller, select the Plate Queue tab, click Add Plates and select the plates to add to the queue. When the queue is complete, click Start Batch. When the run is completed, proceed to Analyze the experiment. c. Click Open/Close to rotate the instrument tray to the OUT position. d. Verify that the associated plate document is open in the SDS software. e. Put the prepared reaction plate in the instrument tray so that: Well A1 is at the top left corner of the tray and the notched corner is at the top right. The bar code is toward the front of the instrument. f. Click Start Run. The instrument tray rotates to the IN position. During the run, the instrument displays real-time status information in the Instrument Real-Time tabs as an amplification plot (which shows the overall fluorescence vs. completed cycles) or as a temperature plot (showing temperatures as a function of time during the run). g. When the Run Complete dialog box appears, click OK to close the dialog box, click Open/Close, then remove the TaqMan Array card from the instrument tray. Analyze the experiment The comparative C T (ΔΔC T ) method uses arithmetic formulas to determine change in expression of a target in an experimental sample relative to the same target in a reference sample. The ΔΔC T method is used for high-throughput measurements of relative gene expression when many gene targets are measured in each sample. For more information on how to analyze ΔΔC T experiments and set up an RQ Study, refer to the Applied Biosystems 7900HT Fast Real-Time PCR System Relative Quantitation Using Comparative CT Getting Started Guide (PN ). Review the chapter on analyzing and viewing RQ Study data in the RQ Manager. Brief procedures for reviewing results and analyzing data are provided below. Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin 7

8 Related information Review the data 1. Transfer the SDS plate document file (*.sds) into an RQ Study, then analyze the study. For optimal results, we recommend the following: For Applied Biosystems TaqMan master mixes, analyze the study with Automatic Baseline and Manual C T set to 0.2. View the amplification plot, then review the baseline and threshold settings. If needed, adjust the baseline and threshold settings for individual assays. IMPORTANT! The same threshold setting must be used for an assay across all samples or TaqMan Array cards within a study. 2. In the well table or results table, review the C T values for each well and for each replicate group. If needed, omit outliers. 3. Review the gene expression plot in the Plate, Detector, or Sample view. Perform downstream analysis For additional analysis, the raw C T values can be exported as a text file (*.txt) then opened in a spreadsheet application (for example, Microsoft Excel). If you use SDS software v2.3 or later, you can export the raw C T values from the Plate Centric view in the RQ Study. For detailed downstream analysis, we recommend: DataAssist, a free software package that can be downloaded from Real-Time StatMiner software for detailed statistical analysis. For information, refer to: Related information System requirements Instrument setup Applied Biosystems 7900HT Fast Real-Time PCR System or ABI PRISM 7900HT Sequence Detection System Sequence Detection Systems (SDS) Software v2.1 or later Sorvall, Heraeus SL40, or Rotanta centrifuge 7900HT TaqMan Array Upgrade Kit (PN ) Make sure that: Your laboratory conforms to the Safety and EMC Compliance guidelines in the Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation and Safety Guide (PN ). SDS Software v2.1 or later is installed on the computer. A TaqMan Array Thermal Cycling Block is installed in the instrument. A background run and pure dye runs have been performed and instrument performance has been verified within the last 6 months. For more information, refer to the SDS Software Online Help. 8 Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin

9 Performing Rapid Cycling Gene Quantitation on TaqMan Array Microfluidic Cards Related information Materials not provided Product Part Number TaqMan Custom Arrays TaqMan Array Gene Signature Panels Go to TaqMan Universal PCR Master Mix, No 1 50-mL tube: AmpErase UNG 10 5-mL tubes: mL tubes: mL tubes: mL tube: TaqMan Universal Master Mix II, No UNG 1 50-mL tube: mL tubes: mL tubes: mL tubes: mL tube: mL tube: TaqMan Array Human MicroRNA Cards A+B Cards Set v3.0, 8 pack: B Cards v3.0, 4 pack: Set Cards v2.0, 8 pack: A Cards v2.0, 4 pack: B Cards v2.0, 4 pack: TaqMan Rodent MicroRNA Cards A+B Cards Set v2.0, 8 pack: A Cards v2.0, 4 pack: B Cards v2.0, 4 pack: TaqMan PreAmp Master Mix Master Mix: Master Mix Kit: TaqMan PreAmp Pools for Human Stem Cell Pluripotency Array: for Mouse Stem Cell Pluripotency Array: for Human GPCR Array: for Rat GPCR Array: TaqMan PreAmp Cells-to-CT TaqMan PreAmp Cells-to-CT Kit with Manual: M TaqMan PreAmp Cells-to-CT Kit: Cells-to-CT Stop Solution: Cells-to-CT Bulk Lysis Reagents: C Applied Biosystems TaqMan Array Microfluidic Cards User Bulletin 9

10 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. NOTICE TO PURCHASER: PLEASE REFER TO THE TAQMAN ARRAY MICRO FLUIDIC CARDS USER GUIDE FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. Purchase of this instrument does not convey any right to practice the 5 nuclease assay or any of the other real-time methods covered by patents owned by Roche or Applied Biosystems. TRADEMARKS: Trademarks mentioned herein are the sole property of Life Technologies Corporation or their respective owners. AmpErase and TaqMan are registered trademarks of Roche Molecular Systems, Inc. RealTime StatMiner is a trademark of Integromics. Sorvall is a trademark of Thermo Fisher Scientific. Copyright 2010, Life Technologies Corporation. All rights reserved. Part Number Rev. A 07/2010 Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone Technical Resources and Support For the latest technical resources and support information for all locations, please refer to our Web site at