Thermo Fisher Scientific GeneChip High Throughput Pico Kit Assay 96-Array Format Automated Workflow User guide

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

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Biomek is a registered trademark of Beckman Coulter,

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

GmbH. Costar is a registered trademark of Corning, Inc. FrameStar is a registered trademark of 4titude Ltd.

Thermo Fisher Scientific Baltics UAB

V.A. Graiciuno 8, LT-02241

Vilnius, Lithuania

Products: GeneChip™ High Throughput Pico Kit

Affymetrix Pte Ltd

7 Gul Circle #2M-01

Keppel Logistics Building

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’

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’

NNN

T7 Promoter

PCR

T7 Promoter

NNN

IVT Pico Amplication and Labeling Process Assay Workow

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

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 (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

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

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Thermo Fisher Scientific GeneChip High Throughput Pico Kit Assay 96-Array Format Automated Workflow User guide

Thermo Fisher Scientific GeneChip High Throughput Pico Kit Assay 96-Array Format Automated Workflow User guide

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