MicroRNA Profiling with TaqMan OpenArray Plates
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1 MicroRNA Profiling with TaqMan OpenArray Plates 1 12/4/2012
2 Profiling with TaqMan MicroRNA Assays Highly Parallel Assays For mirna Expression Analysis Megaplex Primer Pools TaqMan OpenArray mirna Panel OpenArray Instrumentation 2 12/4/2012
3 AB Assay Objective Precursor mirna Mature mirna Develop a Real-Time PCR TaqMan Assay to quantify mature* micrornas *Mature mirnas are the biologically active form 3 12/4/2012
4 TaqMan MicroRNA Assays Design 4 12/4/2012
5 MicroRNA QuantStudio 12K Flex Panels A new standard in high-throughput profiling What are they? Research use, fixed-content OpenArray MicroRNA Panels containing validated TaqMan MicroRNA Assays* for Human and Rodent (Mouse and Rat) Where did they come from? Sanger mirbase release v.14.0 (Human) and release v.15.0 (Rodent) What will they do? Profile well-characterized and well-studied mirnas for Cancer biomarker discovery research, MicroRNA regulation of mrna gene expression, and MicroRNA functional analysis Performance >95% of test assays demonstrating StDev <0.5 among replicates = 5 12/4/2012
6 OpenArray plate Anatomy OpenArray plate = a microscope slide sized plate containing 3072 through-holes 3,072 THROUGH-HOLES divided into smaller groups of 8x8 = SUBARRAYS 48 SUBARRAYS Hydrophobic Hydrophilic 33 nl volume 6 12/4/2012
7 What is an OpenArray plate? Steel plate containing 4 x 12 subarrays comprised of 8 x 8 through-holes for a total of 3072 through-holes. Each through-hole hosts a single RT-PCR reaction Each through-hole can be compared to a well in a conventional plate A B C D OpenArray plate ABC12 a b c d e f g subarray Through-hole B7e8 h /4/2012
8 MicroRNA QuantStudio 12K Flex Panels Broad coverage Run three samples per OpenArray plate 818 assays per sample (758 unique assays) > 3 positive and 1 negative/process control per subarray High-throughput 12 samples per ~2.5 hr instrument run 48 samples per 8hr working day Cost effective Rapid sample screening with minimal hands-on time Minimal sample requirement 100ng of total RNA recommended for a full profile Pool B Sample 3 Pool A Pool B Sample 2 Pool A Pool B Sample 1 Pool A 8 12/4/2012
9 OpenArray mirna Profiling Panels OpenArray Human MicroRNA Panel QuantStudio 12K Flex Minimum order size 1 panel Inventoried 2-3 days shipping P/N Sample 1 Sample 2 Sample 3 OpenArray Rodent MicroRNA Panel QuantStudio 12K Flex Minimum order size 1 panel Inventoried 2-3 days shipping P/N Sample 1 Sample 2 Sample /4/2012
10 OpenArray mirna Profiling Workflow Megaplex RT Primer Pool A Megaplex Preamp Primer Pool A TaqMan OpenArray mirna Panel Reverse Transcription TaqMan MicroRNA RT Kit Preamplification TaqMan PreAmp MMx qpcr TaqMan OpenArray Real-Time MMx Sample 1 Sample 2 Sample 1 Total RNA Sample 384-well Sample Plate Sample 3 3 Samples 750 Targets each Megaplex RT Primer Pool B Megaplex Preamp Primer Pool B 150 min. 90 min. 30 min. Total time < 8 hours 150 min.* (*up to 4 arrays/12 samples run simultaneously) 10 12/4/2012
11 OpenArray Block User Workflow Overview At Life Technologies TaqMan assays ordered on-line The assays are spotted on the OpenArray plate OpenArray plates staked in case At the Researcher s Lab Load your samples with Master Mix onto the OpenArray plate Place lid on to array case, load on to carrier Cycle and image up to 4 OpenArray plates Results! 11 12/4/2012
12 Gene Expression/miRNA on QuantStudio 12K Flex OpenArray Block 9:00AM 11:00AM 1:00PM 3:00PM 5:00PM 0:20 2:00 Load Run (4 x 2,688) Load Run (4 x 2,688) Load Run (4 x 2,688) Run up to 4 arrays at once! 16 arrays/ day >43,000 reactions, 1 operator Load Run (4 x 2,688) 12 12/4/2012
13 Sample Prep The starting material is total RNA or cell lysate. 50 to 200 ng of input total RNA. For most tissues, 100 ng of total RNA produces a comprehensive microrna profile with preamplification. RT and pre-amp OpenArray sample+pcr Master mix OpenArray loading, Pool A and pool B 13 12/4/2012
14 Step 1. RT Prepare the RT Reaction Mix 1. Thaw the following on ice: Megaplex RT Primers TaqMan MicroRNA Reverse Transcription Kit components MgCl2 (supplied with the Megaplex RT Primers) 2. Combine the following in each of two 1.5-mL microcentrifuge tubes (one for Pool A, the other for Pool B) + 3 ul of total RNA (100 ng) 14 12/4/2012
15 Step 1 : Megaplex RT reaction: 96 well plate Sample 1 33ngs/ul and 6uls Sample 2 33ngs/ul and 6uls Sample 3 33ngs/ul and 6uls 3uls 3uls 3uls 3uls 3uls 3uls uls RT 4.5uls RT mix A mix B uls RT 4.5uls RT mix A mix B uls RT 4.5uls RT mix A mix B A B A B A B 96 well plate RT reaction ~2.5h 15 12/4/2012
16 2. Pre-amplification with Megaplex pools A and B The preamplification reaction has a final volume of 25 μl and contains: 2.5 μl RT product 22.5 μl PreAmp reaction mix, pool A in one tube and pool B in another 16 12/4/2012
17 For sample 1 prepare pre-amp mmix A and B Step 2 : Pre-amplification For sample 2 prepare pre-amp mmix A and B For sample 3 prepare pre-amp mmix A and B 22.5 uls 22.5 uls add 2.5 uls RT 2.5 uls RT reaction A reaction B from Step 1 from Step uls 22.5 uls 22.5 uls 22.5 uls add add 2.5 uls RT 2.5 uls RT 2.5 uls RT 2.5 uls RT reaction A reaction B reaction A reaction B from Step 1 from Step 1 from Step 1 from Step 1 A B A B A B 96 well plate (2 nd ) Pre-amp reaction ~1.5h 17 12/4/2012
18 Step 3 :Dilute the Pre-amplification Product A B A B A B 96 well plate from previous step 4µL + 156µL Preamplified cdna 0.1x TE ph 8.0 A B A B A B 96 well plate (3 nd ) 18 12/4/2012
19 4. Prepare OpenArray sample plate To prepare OpenArray sample plate you need: PCR master mix in 1.5 ml tube aliquots of diluted pre-amp pool A and pool B products 1. Aliquot 22.5 μl of TaqMan OpenArray reaction mix into each of the 2 wells per sample of a clean 96-well plate. 2. For each sample, add 22.5 μl of diluted Pool A PreAmp and 22.5 μl of diluted Pool B PreAmp into the other well. 3. Transfer 5 µl of PCR mix in sample plate /4/2012
20 Step 4: prepare PCR reaction Prepare 22.5uls of PCR Mmix for Sample 1A and 1B Prepare 22.5uls of PCR Mmix for Sample 2A and 2B Prepare 22.5uls of PCR Mmix for Sample 3A and 3B 22.5 uls of diluted preamplified sample from step 3 add 22.5 uls of diluted preamplified sample from step uls of diluted preamplified sample from step 3 add 22.5 uls of diluted preamplified sample from step uls of diluted preamplified sample from step 3 add 22.5 uls of diluted preamplified sample from step 3 A B A B A B 96 well plate (4th) 20 12/4/2012
21 Plate Loading 96 Scheme well plate from previous step A B A B A B 5uls in each well A B A B A B You MUSTuse a TaqMan OpenArray 384-Well Sample Plate 21 12/4/2012
22 45uls in each well Plate Loading Scheme sample 2, sample 3.. TaqMan OpenArray 384-Well Sample plate 5µls in each well 22 12/4/2012
23 Plate area 1 Plate area 4 Plate area 4 A1 Plate area 1 Sample 1 Sample 2 Sample 3 Pool A Pool B Pool A Pool B Pool A Pool B Each subarray overlaps perfectly with a single well in the 384 well sample plate D /4/2012
24 SampleTracker 24 12/4/2012
25 Sample Tracker New utility to map 96-well plates with DNA onto 384-well sample plates or OpenArray plates Launch from instrument home screen Also a standalone application Creates 384-well DNA sample plate and OpenArray plate.csv files for all assay formats of genotyping and gene expression OpenArray plates For fixed spacing 12 tip multichannel pipettes Exports plate maps in.pdf or Excel format /4/2012
26 Sample Tracker Properties Under Properties menu - select experiment type and assay layout Genotyping Gene Expression 26 12/4/2012
27 Sample Tracker - Samples Under Samples menu -import 96-well plate.csv files Input.csv file format for 96-well plates: Files are created by user SamplePlate. SampleInfo. SampleInfo. SerialNumber Address SampleID 1 A01 S101 1 A02 S102 1 A03 S103 SampleInfo. Description SampleInfo. Dilution SamplePlateSelectedArea. SelectedAreaIndex 27 12/4/2012
28 Sample Tracker - Translation The Translation menu shows mapping of 96-well plates into the 384-well sample plate when the 384 Plate tab is selected. The 384-well sample plate.csv file is exported from here for upload to AccuFill System (Sample Integration) 28 12/4/2012
29 Sample Tracker - Translation The Translation menu shows mapping of 96-well plates into the 384-well sample plate when the 384 Plate tab is selected. The 384-well.csv file can be exported and placed in: C:\OpenArray\OpenArray Plates for integration with TPF/SPF file in AccuFill System software /4/2012
30 Sample Tracker - Translation The OpenArray tab shows a map of how the color-coded 96-well plates are loaded on the OpenArray plates. Zoom in to see sample names 30 12/4/2012
31 Sample Tracker Translation Features The 96-well plates can be swapped. The plate maps printed using PDF Plate maps can be exported to AccuFill System or Data collection softwares Normal sample file (96w csv format) AccuFill System Software sample/spf integration (384W format csv) QuantStudio Software sample import (OpenArray format csv) 31 12/4/2012
32 Software Workflow Basics Import sample name file & track to AccuFill System or OpenArray Integrate samples with OpenArray Plate Run the OpenArray Plate Analyze Plates in Study SampleTracker AccuFill Software QuantStudio 12K Flex Software Genotyper v1.2 ExpressionSuite v1.0 DigitalSuite.csv.tpf/.spf.eds.las.edt 32 12/4/2012
33 File Formats.csv Sample file name.tpf/.spf Unique OpenArray file containing Assay IDs/names (similar to.txt set up file ).tpf = Genex assays.spf = genotyping assays.eds.edt Experiment file Template file Embedded thermal protocol info.las Study file 33 12/4/2012
34 In AccuFill System software, v1.x Integrate 384W sample plate + tpf.spf 34 12/4/2012
35 Starting OpenArray Run in QuantStudio 12K Flex Software 1. From the home screen under the Run menu click OpenArray, Get Plate ID 2. Browse for file in Experiment File Location field 3. Can start run using eds, edt, SPF/TPF 35 12/4/2012
36 In Summary: Run Loaded_OpenArray tpf/spf Sample Tracker v1.0.csv AccuFill Software.tpf/.spf QuantStudio 12K Flex Software.eds Genotyper Softwarev1.2 ExpressionSuite Software v1.0.las samples assays data study Track Samples from 96W to 384W or OpenArray formats Integrate and load samples on to OpenArray plate files Loaded_OpenArray Analyze multiple.eds files -transfer by.las 36 12/4/2012
37 Why are we doing this? Samples / sample names get transferred, as follows. We need to be able to track them! 96-well plate: longterm sample storage 384-well plate:used by AccuFill System to transfer samples to OpenArray plate 3072-well OpenArray plate: for cycling and endpoint read QuantStudio Software: data analysis 37 12/4/2012
38 Now it s time to load the Sample Plate OpenArray 384-well SAMPLE plate are used to mix sample with TaqMan OpenArray Real-Time PCR Master Mix centrifuge the plate at 1000 rpm, 1 minute Using a black Sharpie, label the sample plate 12 x 4 sections (= area) Each section contains individual samples 38 12/4/2012
39 Sample Loading using the OpenArray AccuFill System Prepare 384-well sample plate Load tip boxes, OpenArray & Sample plate Choose program and press start Remove loaded OpenArray Plate 39 12/4/2012
40 Load tip boxes, OpenArray & Sample plate 40 12/4/2012
41 OpenArray plate guidelines Thaw plates for at least 15 minutesat RT before opening the sealed pouches. Once you remove them, proceed to The AccuFill System. Plates need to be loaded within one hourof opening. Handle cases with snug, powder-free gloves. Only touch OpenArray plates on the case edges. Be careful not to touch the through-holes! If you drop an OpenArray plate, discard it /4/2012
42 Additional preparations While OpenArray plates are thawing in their pouches, prepare immersion fluid syringes. Simply remove cap, attach the tip, and set on a clean surface /4/2012
43 Additional preparation Using a razor blade, score the foil covering the 384- well plate around the area to be loaded. Sections needed 43 12/4/2012
44 Load plates, remove foil Load the 384-Well OpenArray Sample Plate and the OpenArray reaction plate(s) into the AccuFill System. Using tweezers or forceps, remove foil from the Sample Plate /4/2012
45 Affix case lids to OpenArray plate Take an OpenArray Case Lid. Using tweezers or forceps, remove the protective film from the adhesive strip (on one side) and the glass cover (other side) /4/2012
46 Seal OpenArray Plate Set the first OpenArray reaction plate into the Plate Press, as shown: Set the Lid on top Up and to the left 46 12/4/2012
47 Seal OpenArray Plate Press down firmly for exactly 10 seconds. Not too firmly, though /4/2012
48 Immersion fluid Slowlyinject fluid from the syringe into the fill port of the sealed case /4/2012
49 Plug the case Insert a plug, and twist clockwise until tight. Then, remove knob. Plug Hold vertically 49 12/4/2012
50 Clean Loaded Case Wet a lab wipe with EtOH. Wipe the case clean, being carefulnot to press too hard on the sides. Dry with a clean wipe. Change your gloves if immersion fluid on it 50 12/4/2012
51 Load into the Instrument Load 1-4 OpenArray loaded and sealed plates into the QuantStudio 12K Flex real-time instrument using the dedicated carrier. Use correct experimentspecific template(s), and start run Run plates lates after 2 hours of loading 51 12/4/2012
52 MicroRNA Data Analyzed using ExpressionSuite 52 12/4/2012
53 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Excel is a trademark of Microsoft Corporation. Sharpie is a registered trademark of Sanford L.P For Research Use Only. Not for use in diagnostic procedures. The figures and illustrations contained in this presentation are for illustrative purposes only. Life Technologies makes no representations or warranties of any kind, express or implied, regarding the content of the figures or illustrations contained in this presentation. NOTICE TO PURCHASER: Limited Use Label License 53 12/4/2012
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