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For Research Use Only. Not for use in diagnostic procedures.
GeneChip™ High Throughput Pico Kit Assay
96-Array Format Automated Workflow
USER GUIDE
for use with:
GeneChip™ Expression Arrays
GeneChip™ High Throughput Pico Kit
Biomek® FXP Target Prep Express System
Catalog Numbers 902791
Publication Number MAN0027955
Revision A.0
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE,
MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history
Important Licensing Information
This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited
Use Label Licenses.
Corporate entity
Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288
TRADEMARKS
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Inc. Bio-Rad, Hard-Shell, and Microseal are registered trademark of Bio-Rad Laboratories, Inc. Greiner Bio-One is a registered trademark of Greiner Bio-One
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Products: GeneChip™ High Throughput Pico Kit
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Singapore 629563
Products: GeneChip™ expression arrays
Table 1 Revision history of Pub. No. MAN0027955
Revision Date Description
A.0 25 January 2023 Initial release in Thermo Fisher Scientific document control system.
Supersedes Affymetrix publication number 703398.
Greiner Bio-One UV Star plater replaced with Corning 96-well UV plate.
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 3
Contents
CHAPTER 1 GeneChip™ High Throughput Pico Kit . . . . . . . . . . . . . . . 8
Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Purpose of the product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Sample requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Performance specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Assay workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
CHAPTER 2 Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . 15
Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Consumables and labware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Consumables and labware images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Labware templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Reagent and labware requirements (Part 1 to Part 6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Reagent and labware required for Part 1—First-Strand cDNA Synthesis, Cleanup, 3'
Adaptor cDNA Synthesis, Pre-IVT Amplification, and cRNA Amplification . . . . . . . . . . 27
Reagent and labware required for Part 2—cRNA Purification . . . . . . . . . . . . . . . . . . . . 28
Reagent and labware required for Part 3—2nd-Cycle ds-cDNA Synthesis and RNA
Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Reagent and labware required for Part 4—ds-cDNA Purification . . . . . . . . . . . . . . . . . . 30
Reagents and labware required for Part 5—ds-cDNA Fragmentation and Labeling and
Hybridization Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Reagent and labware required for Part 6—GeneTitan™ Preparation . . . . . . . . . . . . . . . . . 35
CHAPTER 3 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Procedural notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Implement a plan to maintain procedural consistency . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
RNase contamination prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
DNA contamination prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Contents
4GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
Prepare Control RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
HeLa Control RNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Poly-A RNA Control preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Prepare Total RNA/Poly-A RNA Control Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Preparation of Total RNA plates for processing on automated systems . . . . . . . . . . . . . 41
CHAPTER 4 Biomek® FXP Target Prep Express setup and target
preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Preferences and options in the HT Pico v1.0 method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Method preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Method parameter options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Labware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Part 1—First-Strand cDNA Synthesis, Cleanup, 3' Adaptor cDNA Synthesis, Pre-IVT
Amplification, and cRNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Approximate duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Reagents and labware required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Clean the Auto-Sealing Microplate Lid with Microseal P Pad . . . . . . . . . . . . . . . . . . . . . 49
Prepare the Biomek® FXP TPE System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Breaking the light curtain while running the assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Thaw and prepare the samples and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Start the Biomek Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Begin Part 1—First-Strand cDNA Synthesis, Cleanup, 3' Adaptor cDNA Synthesis,
Pre-IVT Amplification, and cRNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Part 2—cRNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Approximate duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Reagents and labware required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Perform a pre-run check of the Biomek FXP TPE System . . . . . . . . . . . . . . . . . . . . . . . . . 64
Prepare the samples and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Begin Part 2—cRNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Part 3—2nd-Cycle ds-cDNA Synthesis and RNA Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . 81
Approximate duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Reagents and labware required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Perform a pre-run check of the Biomek FXP TPE System . . . . . . . . . . . . . . . . . . . . . . . . . 82
Thaw and prepare the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Begin Part 3—2nd-Cycle ds-cDNA Synthesis and RNA Hydrolysis . . . . . . . . . . . . . . . . . 83
Part 4—ds-cDNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Approximate duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Reagents and labware required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Perform a pre-run check of the Biomek FXP TPE System . . . . . . . . . . . . . . . . . . . . . . . . . 91
Thaw and prepare the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Contents
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 5
Begin Part 4—ds-cDNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Part 5—ds-cDNA Fragmentation and Labeling and Hybridization Setup . . . . . . . . . . . . . . 107
Approximate duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Reagents and labware required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Perform a pre-run check of the Biomek FXP TPE System . . . . . . . . . . . . . . . . . . . . . . . . 111
Thaw and prepare the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Begin Part 5—ds-cDNA Fragmentation and Labeling and Hybridization Setup . . . . . . . 111
Part 6—GeneTitan™ Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Approximate duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Reagents and labware required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Perform a pre-run check of the Biomek FXP TPE System . . . . . . . . . . . . . . . . . . . . . . . . 129
Labeling GeneTitan™ reagent trays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Thaw and prepare the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Anti-static procedure for GeneTitan™ Instrument trays and covers . . . . . . . . . . . . . . . . . 131
Begin Part 6—Preparation for the GeneTitan™ Instrument . . . . . . . . . . . . . . . . . . . . . . . 133
APPENDIX A GeneChip™ HT Pico workflow diagram . . . . . . . . . . . . 139
First-Strand cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3’ Adaptor cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Pre-IVT Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
cRNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
cRNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
cRNA Quantitation and Normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
2nd-Cycle ds-cDNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
RNA Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
ds-cDNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
ds-cDNA Quantitation and Normalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
ds-cDNA Fragmentation and Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
3’ IVT cartridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
WT cartridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Plate arrays and Clariom™ S cartridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Hybridization Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3’ IVT Cartridge Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
WT Cartridge Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Clariom™ S Cartridge Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3’ IVT Array Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
WT and Clariom™ S Array Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
GeneTitan™ Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Contents
6GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
APPENDIX B GeneChip™ HT Pico labware and reagent deck setup
screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Part 1—First-Strand cDNA Synthesis, Cleanup, 3' Adaptor cDNA Synthesis, Pre-IVT
Amplification, and cRNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Part 2—cRNA Purification using plate reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Part 2—cRNA Purification using the NanoDrop™ option . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Part 3—2nd-Cycle ds-cDNA Synthesis and RNA Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . 177
Part 4—ds-cDNA Purification using plate reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Part 4—ds-cDNA Purification using the NanoDrop™ option . . . . . . . . . . . . . . . . . . . . . . . . 179
Part 5—Fragmentation & Labeling and Hybridization Setup: 3' IVT cartridge arrays . . . . 180
Part 5—Fragmentation & Labeling and Hybridization Setup: WT cartridge arrays . . . . . . 181
Part 5—Fragmentation & Labeling and Hybridization Setup:
Clariom™ S cartridge arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Part 5—Fragmentation & Labeling and Hybridization Setup: 3' IVT array plates . . . . . . . . 183
Part 5—Fragmentation & Labeling and Hybridization Setup: WT and
Clariom™ S array plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Part 6—GeneTitan™ Preparation: 3' IVT array plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Part 6—GeneTitan™ Preparation: WT and Clariom™ S array plates . . . . . . . . . . . . . . . . . . 186
APPENDIX C GeneChip™ HT Pico deck layout setup and cleanup . 187
Part 1—First Strand cDNA Synthesis, Cleanup, 3’ Adaptor cDNA Synthesis, Pre-IVT
Amplification, and cRNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Part 2—cRNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Part 3—2nd-Cycle ds-cDNA Synthesis and RNA Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . 190
Part 4—ds-cDNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Part 5—ds-cDNA Fragmentation & Labeling, and Hybridization Setup: cartridge arrays . . 192
Part 5—ds-cDNA Fragmentation & Labeling and Hybridization Setup: WT or
Clariom™ S Array Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Part 5—ds-cDNA Fragmentation & Labeling and Hybridization Setup: 3’ IVT Array Plates 194
Part 6—GeneTitan™ Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Contents
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 7
APPENDIX E Documentation and support . . . . . . . . . . . . . . . . . . . . 199
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
8GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
1GeneChip™ High Throughput
Pico Kit
Product information
Purpose of the
product
The Applied Biosystems GeneChip™ High Throughput (HT) Pico Kit (96 automated
reactions; Cat. No. 902791) and the Pico Kit (30 manual reactions or 24 automated
reactions; Cat. No. 902790) enable you to use automation to prepare hybridization
ready target from picogram to nanogram quantities of total RNA samples for gene
expression profiling analysis with gene-level expression arrays such as 3' IVT, Gene ST,
and Clariom™S, among many others (Figure 1). Reverse transcription is initiated at the
poly-A tail as well as throughout the entire length of RNA to capture both coding and
multiple forms of non-coding RNA, making the Pico Kit ideal for amplification of
intact, partially degraded, and compromised RNA samples. Complementary RNA
(cRNA) amplification is achieved using low-cycle PCR followed by linear
amplification using T7 in vitro transcription (IVT) technology. The cRNA is then
converted to biotinylated double-stranded cDNA (ds-cDNA) hybridization targets for
unbiased coverage of the transcriptome. The kit is optimized to work with a wide
range of samples including tissues, cell lines, whole blood, and formalin-fixed
paraffin-embedded (FFPE) tissues.
The High Throughput Pico v1.0 method is for use only with Biomek Software version
4.1 (Windows 7) controlling the Beckman Coulter Biomek® FXP Target Prep Express
(TPE) System. HT Pico automated target preparation encompasses all assay stages,
beginning with cDNA synthesis from RNA samples and finishing with either
hybridization ready target and stains in GeneTitan™ trays or hybridization ready
target for cartridge arrays in a 96 well FrameStar Break-A-Way PCR plate.
The Beckman Coulter Biomek FXP TPE System automates the preparation of target that
can be hybridized to a:
• GeneChip™ cartridge array that can be processed using the Hybridization Oven
640/645, GeneChip Fluidics Station FS450, and GCS 3000 Scanner System
Target preparation for cartridge arrays uses the first 5 parts of the Biomek FXP
method with an optional stopping point at the completion of each part and at
user-selectable ‘Stop Steps’ (Figure 1). At the end of target preparation, the
biotinylated double-stranded cDNA target is ready to be denatured and
hybridized onto a gene-level expression cartridge array such as 3' IVT, Gene ST,
and Clariom S, among many others.
• GeneChip array plate that can be processed using the GeneTitan™ Instrument
NOTE: Thermo Fisher Scientific does not recommend the use of user prepared
reagents for the HT Pico Kit. Use the GeneTitan™ Hybridization, Wash, and Stain Kit
for WT Array Plates (Cat. No. 901622), 3' IVT Array Plates (Cat. No. 901530) or the
GeneChip™ Hybridization, Wash, and Stain Kit (cartridge arrays, Cat. No. 900720) for
all the reagents needed for the hybridization, wash, and stain steps of this assay.
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 9
Chapter 1 GeneChip™ High Throughput Pico Kit
Product information 1
Target preparation for array plates uses all 6 parts of the Biomek FXP method with
an optional stopping point at the completion of each part and at user-selectable
‘Stop Steps’. At the end of target preparation, the denatured biotinylated double-
stranded cDNA target is ready to be hybridized onto a gene-level expression
array plate such as 3' IVT, Gene ST, and Clariom S, among many others.
Sample
requirements
The Pico Kits and HT Pico v1.0 method together comprise reagents and a protocol for
producing hybridization-ready DNA on a Biomek FXP TPE System starting with
100 pg to 10 ng of purified total RNA from cells or tissues or with 500 pg to 50 ng of
purified total RNA from FFPE tissues. The total RNA samples can be used directly
without removal of ribosomal or globin RNA prior to target preparation with the Pico
Kit. The recommended total RNA inputs in Table 1 are based on total RNA from HeLa
cells and 1 to 9 year old FFPE tissues.
To ensure high reproducibility in whole transcriptome amplification, we recommend
using a starting amount of no less than 500 pg RNA template of purified total RNA
from cells or tissues and 2 ng of purified total RNA from FFPE tissues. Depending on
the copy number of the transcripts, it may be possible to use smaller amounts of RNA
template (Table 1). Input amounts that are lower than the recommended amounts can
result in insufficient yields, poor conversion to ds-cDNA, and reduction in array
signals. The starting RNA template should not be less than the minimum input
amounts listed in Table 1. If your RNA sample is not limiting, we recommend that you
start with more total RNA.
Performance
specifications
The HT Pico Kit and protocol has been verified to generate greater than 20 µg cRNA
and greater than 11 µg of ds-cDNA from either 500 pg of HeLa total RNA or 2 ng of
1 to 9 years old FFPE tissues. This should provide sufficient biotin-labeled ds-cDNA
target from each sample for hybridization to a single GeneChip Expression Array. For
information on RNA preparation, see Chapter 3, "Preparation" on page 36.
Table 2 to Table 7 provide the time required to perform each part of the assay.
Depending on the selected parameters, target preparation takes about 27 hours and
requires 8 user interventions. Additional time is required for hybridization, washing,
staining, and scanning, and is dependent on the number of samples processed and the
array type.
This manual describes the assay procedures recommended for target labeling for
whole transcriptome expression analysis using the Beckman Coulter Biomek FXP TPE
System. The reagents and protocols have been developed and optimized specifically
for use with the Beckman Coulter Biomek FXP TPE System.
Table 1 Input RNA limits.
RNA Input Total RNA from fresh-
frozen cells or tissues
Total RNA from formalin-fixed,
paraffin-embedded tissues
Minimum 100 pg 500 pg
Recommended 500 pg to 10 ng 2 ng to 50 ng
Maximum 10 ng 50 ng
NOTE: Complementary RNA (cRNA) is also known as amplified RNA (aRNA).
Chapter 1 GeneChip™ High Throughput Pico Kit
Product information
10 GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
1
Assay workflow
Figure 1 Overview of the high throughput Pico amplification and labeling process.
3’ Adaptor Synthesis
40 min
3’
cRNA Amplification
14 hr
Fragmentation and Terminal Labeling
1 hr 5 min
Stop Step
2nd-Cycle ds-cDNA Synthesis
1 hr 10 min
Template RNA Removal
20 min
Hybridization Setup
Day 1Day 2
AAAAAAAAAAAAAAA 3’
5’
5’
3’
5’ NNN
5’
3’ 3’
5’
dUTP
3’
5’
dUTP
DNA
RNA
NNN Random Primers
TTT dT Primers
DLR DNA Labeling Reagent
Biotin
ds-cDNA Purification and
Quantification
Stop Step
ds-cDNA Normalization
First-Strand cDNA Synthesis
1 hr 5 min
Incubation Time
UDG &APE1
TdT & DLR
Reverse Transcriptase
NNN5’ PCR
Clean-up Excess Primers
40 min
Exonuclease
T7 Promoter 5’
PCR
3’
PCR
T7 Promoter 5’
PCR
Pre-IVT Amplification
< 1.5 hr
Taq
3’
PCR
T7 Promoter 5’
PCR
5’ PCR 3’
PCR
T7 RNA Polymerase
Reverse Transcriptase
DNA Polymerase
Hydrolysis
TTTT
T7 Promoter
PCR
5’
5’
NNN
T7 Promoter
PCR
T7 Promoter
NNN
IVT Pico Amplication and Labeling Process Assay Workow
Exo-free Klenow
5’
3’
cRNA Purification and Quantification
Stop Step
cRNA Normalization
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 11
Chapter 1 GeneChip™ High Throughput Pico Kit
Product information 1
Table 2 Approximate time required for Part 1.
Part 1—First-Strand cDNA synthesis through cRNA Amplification 24X (minutes) 96X (minutes)
Deck setup 15 15
Biomek FXP TPE System pipetting steps: First-strand synthesis 6 8
Thermal cycler: first-strand method 70 70
Biomek FXP TPE System pipetting steps: Cleanup 6.5 12
Thermal cycler: Cleanup method 45 45
Biomek FXP TPE System pipetting steps: 3' Adaptor Synthesis 8 10
Thermal cycler: 3' Adaptor Synthesis 45 45
Biomek FXP TPE System pipetting steps: Pre-IVT Amplification 8 16
Thermal cycler: PCR method (9 cycles) 55 55
Biomek FXP TPE System pipetting steps: cRNA Amplification 7.5 16
Thermal cycler: IVT method 840 840
Part 1 total time 1,106 minutes
18.4 hours
1,132 minutes
18.9 hours
Table 3 Approximate time required for Part 2.
Part 2—cRNA Purification 24X (minutes) 96X (minutes)
Deck setup 15 15
Biomek FXP TPE System pipetting steps: cRNA purification 66 77
Biomek FXP TPE System pipetting steps: cRNA normalization 4 12
Part 2 total time 85 minutes
1.4 hours
104 minutes
1.7 hours
Chapter 1 GeneChip™ High Throughput Pico Kit
Product information
12 GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
1
Table 4 Approximate time required for Part 3.
Part 3—2nd-Cycle ds-cDNA Synthesis 24X (minutes) 96X (minutes)
Deck setup 10 10
Biomek FXP TPE System pipetting steps: 2nd-cycle synthesis 6 13
Thermal cycler: 2nd-cycle synthesis method 75 75
Biomek FXP TPE System pipetting steps: RNA hydrolysis 5 7
Thermal cycler: Hydrolysis method 25 25
Biomek FXP TPE System pipetting steps: Neutralization 4 6
Part 3 total time 125 minutes
2.1 hours
136 minutes
2.3 hours
Table 5 Approximate time required for Part 4.
Part 4—ds-cDNA Purification 24X (minutes) 96X (minutes)
Deck setup 15 15
Biomek FXP TPE System pipetting steps: ds-cDNA purification 64 73
Biomek FXP TPE System pipetting steps: ds-cDNA normalization 4 12
Part 4 total time 83 minutes
1.4 hours
100 minutes
1.7 hours
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 13
Chapter 1 GeneChip™ High Throughput Pico Kit
Product information 1
Table 6 Approximate time required for Part 5.
Part 5—ds-cDNA Fragmentation & Labeling, and Hybridization Setup 24X (minutes) 96X (minutes)
Deck setup 10 10
Biomek FXP TPE System pipetting steps: cDNA fragmentation and labeling 7 9
Thermal cycler: fragmentation and labeling method 65 65
Biomek FXP TPE System pipetting steps: Hybridization setup
GeneChip™ Cartridge Array Hybridization setup
or
GeneChip™ 3’ IVT Array Plate Hybridization setup
or
GeneChip™ WT and Clariom™ S Array Plate Hybridization setup
13
13
28
22
18
28
Part 5 total time—Cartridge Array
Part 5 total time—3’ IVT Array Plate
Part 5 total time—WT and Clariom S Array Plate
95 minutes
1.6 hours
95 minutes
1.6 hours
110 minutes
1.8 hours
106 minutes
1.8 hours
102 minutes
1.7 hours
112 minutes
1.9 hours
Table 7 Approximate time required for Part 6.
Part 6—GeneTitan Preparation 24X (minutes) 96X (minutes)
Deck setup 10 10
Biomek FXP TPE System pipetting steps and thermal cycler: Denaturation
protocol
18 18
Part 6 total time 28 minutes 28 minutes
Chapter 1 GeneChip™ High Throughput Pico Kit
Kit contents and storage
14 GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
1
Kit contents and storage
Table 8 GeneChip™ Pico Kit (30 Rxn, Cat. No. 902790) and HT Pico Kit (96 Rxn, Cat. No. 902791).
Component Storage
Pico Amplification Kit, Module 1, –20°C: cRNA Step
Pico First-Strand Enzyme –20°C
Pico First-Strand Buffer –20°C
Pico Cleanup Reagent –20°C
Pico 3' Adaptor Enzyme –20°C
Pico 3' Adaptor Buffer –20°C
Pico PCR Enzyme –20°C
Pico PCR Buffer –20°C
Pico IVT Enzyme –20°C
Pico IVT Buffer –20°C
Poly-A Control Stock –20°C
Nuclease-free Water any temp1
1 Store the Nuclease-free Water at –20°C, 4° C, or room temperature.
Pico Amplification Kit, Module 2, –20°C: cDNA Step
Pico 2nd-Cycle Primers –20°C
Pico 2nd-Cycle ds-cDNA Enzyme –20°C
Pico 2nd-Cycle ds-cDNA Buffer –20°C
Pico Frag. & Label Enzyme –20°C
Pico Frag. & Label Buffer –20°C
Pico Amplification Kit, Module 3, 4°C
Purification Beads 4°C2
2 Do not freeze.
Pico Hydrolysis Buffer 4°C†
Pico Neutralization Buffer 4°C†
Control RNA, –20°C: HeLa Total RNA
Control RNA (100 ng/µL HeLa total RNA) –20°C
GeneChip™ Hybridization Control Kit
20X Hybridization Controls –20°C
3 nM Control Oligo B2 –20°C
Tubes Organizer: Plastic vinyl template for organization and storage of components in 9x9 array,
81-places square wells, 5 1/4 in. x 5 1/4 in (e.g., Nalgene CryoBox Cat. No. 5026-0909, or
equivalent).
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 15
2Required materials
Equipment, consumables, labware, and reagents required
Unless otherwise indicated, all materials are available through thermofisher.com.
“MLS” indicates that the material is available from fisherscientific.com or another
major laboratory supplier.
Equipment
Table 9 Equipment required for target preparation.
Item Quantity Source
Beckman Coulter Biomek FXP Target Prep Instrument 1 Beckman Coulter, Cat. No. A83103
24-position tube rack with Tube Inserts
(11 mm, white, case of 25)
1 Beckman Coulter
Cat. No. 373661 (rack) and
Cat. No. 373696 (inserts)
Blue Heater/Chiller 24 Well block (referred to as Reagent Cold
block, 24 Well Block in this document)
1 Beckman Coulter, Cat. No. A80354
Adapter, Thin PCR (installed on the Shaking Peltier) 1 Beckman Coulter, Cat. No. A19709
Centrifuge (for tube and plate) 1 MLS
Pipettes 1 MLS
Spectrophotometer (plate, single-channel, or multi-channel) 1 MLS
Thermal Cycler and Adhesive PCR Plate Seals (optional for off-
deck incubation)
1 MLS
Vortex Mixer 1 MLS
Chapter 2 Required materials
Equipment, consumables, labware, and reagents required
16 GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
2
Consumables and labware
Thermo Fisher Scientific does not offer a consumables kit specifically for use with the
HT Pico Biomek v1.0 method. The HT WT PLUS Consumables for Biomek FXP Target
Prep Express (Cat. No. 902508) when supplemented with 2 additional P Pads (Bio-Rad
Cat. No. MSP1003) will be sufficient to perform 4 complete HT Pico assays for cartridge
arrays or 3' IVT array plates. When supplemented with 2 additional P Pads (Bio-Rad
Cat. No. MSP1003) and 4 additional Bio-Rad Hard Shell PCR Plates (Bio-Rad Cat. No.
HSP-9601 or HSP-9631) the kit will be sufficient to perform 4 complete HT Pico assays
for WT or Clariom™ S array plates.
Table 10 Expression installation kits required for HT Pico method.
Item Quantity Source
Applied Biosystems HT 3' IVT PLUS and WT PLUS
Core Installation Kit Module consists of:
• Alpaqua Magnetic Plate
• Alpillo® Plate Cushion
• Arched Auto-Sealing Lids With Wide Tabs
1
1
1 (Pkg of 4)
902453
Applied Biosystems HT Pico Installation Kit consists
of:
• HT Pico Cold Block Template
• HT Pico Reservoir Stickers
• PCR Tube Holder
1
1
1
902901
HT Pico v1.0 Biomek method file (and support files) Installed by
Thermo Fisher
Scientific FAS
Table 11 Equipment required for array processing.
Instrument Source
GeneChip™ System for Cartridge Arrays
• GeneChip™ Hybridization Oven 645
• GeneChip™ Fluidics Station 450
• GeneChip™ Scanner 3000 7G
00-0331 (110/220V)
00-0079
00-0212 (North America)
00-0213 (International)
GeneChip™ AutoLoader with External Barcode Reader 00-0090 (GCS 3000 7G S/N 501)
00-0129 (GCS 3000 7G S/N 502)
GeneChip™ System for Array Plates
• GeneChip™ MC Instrument, NA/Japan includes 110V UPS
• GeneChip™ MC Instrument, Int'l includes 220V UPS
• GeneChip™ Instrument, NA/Japan includes 110V UPS
• GeneChip™ Instrument, Int'l includes 220V UPS
00-0372
00-0373
00-0360
00-0363
GeneTitan™ ZeroStat AntiStatic Gun (for processing array plates) 74-0014
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 17
Chapter 2 Required materials
Equipment, consumables, labware, and reagents required 2
Table 12 Biomek® Tips and universal labware required for 1 full run of the HT Pico Kit on the Biomek Workstation.
Item Packaging Total
24X
Total
96X
Source
Biomek P50 Tips, Pre-sterile with Barrier (magenta)196 ea./box
10 boxes/
case
12 boxes 14 boxes Beckman Coulter,
Cat. No. A21586
Biomek AP96 P250 Tips, Pre-sterile with Barrier
(AP96 Multichannel Pipettor) (aqua)
• Plates
• Cartridges
96 ea./box
10 boxes/
case 6 boxes
5 boxes
6 boxes
5 boxes
Beckman Coulter,
Cat. No. 717253
Biomek Span-8 P250 Tips, Pre-sterile with Barrier (Span
8 Pipettor) (green)
96 ea./box
10 boxes/
case
1 Box 2 boxes Beckman Coulter,
Cat. No. 379503
Biomek Span-8 P1000 Tips, Conductive, Pre-sterile with
Barrier (Span 8 Pipettor) (yellow)2, or
Biomek Span-8 P1000 Tips, Pre-sterile with Barrier
(Span 8 Pipettor) (orange)
96 ea./box
5 boxes/
case
2 boxes 2 boxes Beckman Coulter,
Cat. No. 987925
Reservoir, Quarter, Divided by Width
• Plates
• Cartridges
48 units/
case 6 units
4 units
6 units
4 units
Beckman Coulter,
Cat. No. 372792
Reservoir, Quarter
• Plates
• Cartridges
48 units/
case 5 units
4 units
5 units
4 units
Beckman Coulter,
Cat. No. 372790
Frame for reservoirs 1 ea. 3
Frames
reusable
3 Frames
reusable
Beckman Coulter,
Cat. No. 372795
1 Two fewer boxes of Biomek P50 Tips, Pre-sterile with Barrier (magenta) will be needed if selecting the NanoDrop™ quantitation option
for Parts 2 and 4.
2 HT Pico v1.0 method can use either conductive (yellow tip box) or non conductive (orange tip box) P1000 Span-8 Barrier Pipette tips
interchangeably.
18 GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
Chapter 2 Required materials
Equipment, consumables, labware, and reagents required
2
Table 13 Pipette tip usage for 1 full run of the HT Pico Kit on the Biomek Workstation.
Category Item Total1Part 1 Part 2 Part 3 Part 4 Part 5 Part 62
Cartridge Plate
24X 96X 24X 96X 24X 96X 24X 96X 24X 96X 24X 96X 24X 96X 24X 96X
Pipette tip
box
AP96 P50 Tips
(magenta)
101144113311111200
AP96 P250 Tips
(aqua)
6600220022111111
Span-8 P50
Tips (magenta)
2311111011001000
Span-8 P250
Tips (green)
1211111111111100
Span-8 P1000
Tips (yellow)
2211111111111111
Tip count AP96 P50 Tips
(magenta)
960 1056 384 384 963963288 288 96396396 96 96 192 0 0
AP96 P250 Tips
(aqua)
576 576 0 0 192 192 0 0 192 192 96 96 96 96 96 96
Span-8 P50
Tips (magenta)
144 288 48 96 24 96 16 0 24 96 8 0 32 0 0 0
Span-8 P250
Tips (green)
77 124 19 40 8 8 8 40 8 8 2 8 34 28 0 0
Span-8 P1000
Tips (yellow)
116 131 6 15 32 32 3 4 32 32 22 31 19 24 24 24
1Total for array plates with GeneTitan Hybridization options. Fewer tips are required for cartridge array options.
2Not required for cartridge array options.
3 No AP96 tips are required (magenta) for Part 2 and 4 if using the NanoDrop™ option.
All tips used for the MC pod are to be discarded after the run, but if being used for the Span-8 pod, then the tips can be used for the next Part if continuing (a new box of Span-8 tips is
always required when resuming a run after the method has been stopped.)
GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide 19
Chapter 2 Required materials
Consumables and labware images 2
Reagents
Consumables and labware images
Table 15 shows the types of labware used in the GeneChip HT Pico Assay. For labware
requirements specific to each part of the assay, see "Reagent and labware requirements
(Part 1 to Part 6)" on page 27.
Table 14 Additional reagents required.
Item Source
GeneChip™ Hybridization, Wash, and Stain Kit (30 Rxn, for cartridge arrays) 900720
GeneTitan™ Hybridization, Wash, and Stain Kit for 3' IVT Array (96 Rxn) 901530
GeneTitan™ Hybridization, Wash, and Stain Kit for WT Array Plates (96 Rxn) 901622
DNAZap™ PCR DNA Degradation Solutions (or equivalent product) AM9890
RNaseZap™ RNase Decontamination Wipes (or equivalent product) AM9786
Nuclease-free Water
(for elution, dilution and preparing 80% Ethanol Wash Solution:
~45 mL for 1 × 24 rxn, and ~120 mL for 1 × 96 rxn)
MLS
100% Ethanol (Molecular Biology grade or equivalent)
(for preparing 80% Ethanol Wash Solution:~20 mL for 1 × 24 rxn, and ~50 mL for 1 × 96 rxn)
MLS1
1 Before handling any chemicals, see the SDS provided by the manufacturer, and observe all relevant precautions.
Table 15 Consumables and labware used on the Biomek workstation deck.
Labware Source Labware image
Biomek P50 Pipette Tips
(magenta box; pre-sterile, barrier)
Beckman Coulter
Cat. No. A21586
Biomek AP96 P250 Tips
(aqua box; pre-sterile, barrier)
Beckman Coulter
Cat. No. 717253
Chapter 2 Required materials
Consumables and labware images
20 GeneChip™ High Throughput Pico Kit Assay 96-Array Format Automated Workflow—Biomek® FXP User Guide
2
Biomek Span-8 P250 Tips
(green box; pre-sterile, barrier)
Beckman Coulter
Cat. No. 379503
Biomek Span-8 P1000 Tips
(yellow box; pre-sterile, barrier,
conductive or orange box; pre-
sterile, barrier)
Beckman Coulter
Cat. No. 987925 (yellow)
Cat. No. B01124 (orange)
Reservoir, Quarter
• Quarter module, 40 mL
capacity
• Quarter module divided by
width, 19 mL capacity per
section
Beckman Coulter
Cat. No. 372790 (40 mL)
Cat. No. 372792 (19 mL)
Frame for Reservoirs
(3 total needed for HT Pico
Method, reusable for subsequent
runs)
Beckman Coulter
Cat. No. 372795
96-Well Deep Well Microplate,
Polypropylene, 2 mL, Non-
sterile
A component of the Applied
Biosystems HT WT PLUS
Consumables Kit
Cat. No. 902508, 8 per Kit
(or AbGene Cat. No. AB0932)
Table 15 Consumables and labware used on the Biomek workstation deck. (Continued)
Labware Source Labware image
Undivided
40 mL capacity
Divided by width
19 mL capacity
/
