Multiplex RT for TaqMan Array Human MicroRNA Panel

Transcription

1 f Multiplex RT for TaqMan Low Density Array Multiplex RT for TaqMan Array Human MicroRNA Panel Quick Reference Card For safety and biohazard guidelines, refer to the Safety section in the Multiplex RT for TaqMan MicroRNA Assays Protocol (PN A). For all chemicals in bold red type, read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Multiplex Reverse Transcription IMPORTANT! This procedure assumes that you are quantifying mirna for all wells on a TaqMan Low Density Array. STEP 1 Prepare the RT master mix a. Thaw the TaqMan MicroRNA Reverse Transcription Kit components on ice. b. In a polypropylene tube, prepare the RT master mix for a total of eight multiplex RT reactions (12.5% excess volume is recommended to allow for pipetting losses). c. Mix gently and centrifuge briefly, then place the RT master mix on ice.. Volume (µl) Component For One RT Reaction For One Sample (8 RT Reactions) 100 mm dntps (with dttp) MultiScribe Reverse Transcriptase, 50 U/mL Reverse Transcription (RT) Buffer RNase Inhibitor, 20 U/µL Nuclease-free water Total Includes 12.5% excess volume for pipetting losses. 2 Add the RNA template Dispense 2.0 µl (10 to 100 ng) of total RNA template into each well of a MicroAmp Optical Reaction Plate or each tube of a MicroAmp Optical 8-Tube Strip, then place the plate or tubes on ice.

2 STEP 3 4 Prepare the multiplex RT reaction a. Dispense 7.0 µl of RT master mix into each well of the MicroAmp Optical Reaction Plate or into each Tube. b. Transfer 1 µl of each Multiplex RT Human primer pool into the appropriate wells or tubes. A total of eight independent RT reactions must be run per sample. c. Seal the plate using MicroAmp Optical Adhesive Film, or cap the tubes. d. Mix gently and centrifuge briefly. Do not vortex. Incubate the plate on ice for at least 5 min or until you are ready to load the thermal cycler. Program the thermal cycler Set the thermal cycler to 9600 Emulation mode, then program as follows: Step Type Time (min) Temperature ( C) 5 Perform multiplex reverse transcription HOLD HOLD HOLD 5 85 HOLD 4 Set the volume to 10 µl, load the plate into the thermal cycler, then start the run. Page 2

3 The TaqMan Assay IMPORTANT! For details on working with TaqMan Low Density Arrays, refer to the Applied Biosystems TaqMan Low Density Array User Bulletin (PN ). STEP 1 Prepare the RT reactionspecific PCR mix a. Transfer each RT reaction to a 1.5-mL microcentrifuge tube and dilute 62.5-fold by adding 615 µl of nuclease-free water to each of the eight 10-µL RT reactions. b. For each RT reaction, label a second 1.5-mL microcentrifuge tube. c. Thaw the TaqMan Universal PCR Master Mix. d. Add the following components to each of the second 1.5-mL microcentrifuge tubes: Component Volume (µl) Diluted RT Reaction 50.0 TaqMan Universal PCR Master Mix (2X) 50.0 Total Volume Load the RT reaction-specific PCR reaction mix into fill reservoirs e. Cap the microcentrifuge tubes and mix gently. f. Centrifuge the tubes to eliminate air bubbles. Place on ice. IMPORTANT! After diluting the Multiplex RT reaction, proceed directly to step 2. If necessary, the undiluted Multiplex RT reaction can be stored overnight at 15 to 25 C. Longer storage has not been investigated. a. After the TaqMan Array has equilibrated to room temperature, carefully remove the card from the packaging and place foil-side down on the lab bench. b. Using a 100-µL micropipette, load 100 µl of the RT reaction-specific PCR reaction mix (from step 1 above) into the corresponding fill ports below. Dispense the RT reaction-specific PCR reaction mix so that it seeps in and around the fill reservoir toward the vent port, pipetting the entire 100 µl into the fill reservoir. TaqMan Array Fill Port Multiplex RT Pool IMPORTANT! Do not allow the tip to contact and possibly damage the coated foil beneath the fill port. Page 3

4 STEP 3 Centrifuge the TaqMan Array IMPORTANT! Refer to the Applied Biosystems TaqMan Low Density Array User Bulletin (PN ) for details on centrifuging TaqMan Arrays. Use the following centrifuge settings: Parameter EASYSet (Touchpad) QUICKSet (Knob-operated) 4 Seal the TaqMan Array Up Ramp Rate 9 3 Down Ramp Rate 9 N/A Rotational Speed 1200 rpm (331 g) 1200 rpm Centrifugation Time 2 1 min 2 1 min a. Repeat centrifugation for a total of two consecutive, 1-minute spins to ensure complete distribution of the PCR reaction mix. b. Examine each TaqMan Array to determine if filling is complete. The amount of PCR reaction mix remaining in the fill reservoirs should be uniform and consistent from reservoir to reservoir. IMPORTANT! Refer to the Applied Biosystems TaqMan Low Density Array User Bulletin (PN ) for details on sealing TaqMan Arrays. Using scissors, trim the fill reservoirs from the TaqMan Array. Use the edge of the TaqMan Array carrier as a guide. 5 6 Set up and run the plate document CAUTION! PHYSICAL INJURY HAZARD. Use care when trimming off the fill reservoirs. Use scissors rather than razor blades or other unprotected cutting devices. IMPORTANT! For details on how to configure and run the plate document, refer to the Applied Biosystems TaqMan Low Density Array User Bulletin (PN ) and Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide (PN ). a. Import the SDS setup file from the Array Information CD (shipped with the TaqMan Array) into a new plate document. b. Verify that the TaqMan Array thermal cycling block is installed in the instrument tray. c. Load the TaqMan Array in the correct orientation into the instrument. d. Start the run. Analyze the PCR reactions For information on analyzing the results, see the SDS Online Help, 7900HT System User Guide, or Applied Biosystems 7900HT Fast Real-Time PCR System Relative Quantitation Using Comparative C T Getting Started Guide. Page 4

5 Required Materials Consumables Equipment Item Part Number Item Applied Biosystems 7900HT TaqMan Low Density Array Upgrade Part Number MicroAmp Optical 96-Well Reaction Plate with Barcode (500 plates) MicroAmp Optical 384-Well Reaction Plate with Barcode (50 plates) Reagents MicroAmp Optical Adhesive Film (100 films) Item Part Number TaqMan Multiplex RT for TaqMan MicroRNA Assays Human Multiplex RT Pool 1 v Human Multiplex RT Pool 2 v Human Multiplex RT Pool 3 v Human Multiplex RT Pool 4 v Human Multiplex RT Pool 5 v Human Multiplex RT Pool 6 v Human Multiplex RT Pool 7 v MicroAmp Optical 8-Tube Strip, 0.2-mL (1000 tubes in strips of 8) MicroAmp Optical 8-Cap Strip (300 strips) MicroAmp Cap Installing Tool MicroAmp Adhesive Film Applicator MicroAmp Clear Adhesive Films (100 films) MicroAmp Optical Film Compression Pad (5 pads) Reagent Tubes, 10-mL Human Multiplex RT Pool 8 v Human Multiplex RT Set (Pools 1 8) TaqMan MicroRNA Reverse Transcription Kit (200 reactions) TaqMan MicroRNA Reverse Transcription Kit (1000 reactions) TaqMan 2 Universal PCR Master Mix, No AmpErase UNG TaqMan Low Density Array Human MicroRNA Panel Page 5

6 Notes Copyright 2007, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: PLEASE REFER TO THE PROTOCOL OR PACKAGE INSERT OF THE PRODUCTS NAMED HEREIN FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. Applera, Applied Biosystems, and AB (Design) are registered trademarks, and MicroAmp and MultiScribe are trademarks of Applera Corporation and/or its subsidiaries in the U.S. and/or other countries. TaqMan and AmpErase are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. 05/ Part Number Rev. A