Gene Expression Analysis - Illumina, Inc. · GENE EXPRESSION ANALYSIS sequencing-based gene...

Gene Expression

Analysis

mrna sequencing (mrna-seq)

Overview pages 138–139

reagents pages 140–142

small rna discOvery and analysis

Overview page 143


direct HybridizatiOn gene expressiOn

Overview page 148

Whole-genome expression Kits pages 149–154

WHOle-genOme dasl gene expressiOn


reagents page 157

veracOde dasl gene expressiOn



137

Whether you want to look at entire transcriptomes or only small RNAs, intact RNA

to partially degraded RNA samples, Illumina gene expression solutions can help

you. With high feature redundancy, low sample input requirements, and industry-

leading pricing and performance, you can get more of what you want within the

same budget. Now Illumina gene expression solutions are available on three differ-

ent platforms to match the scale of your research.

Application

mRNA-Seq

Small RNA Discovery and Analysis

Whole-Genome Expression Direct Hybridization

Whole-Genome Analysis of FFPE Samples

Open Platform Assays, Capture Beads (< 384 targets)

Open Platform Assays, Carboxyl Beads (< 384 targets)

Analysis of FFPE Samples (< 384 targets)

An Overview of Illumina’s Gene Expression Solutions

Purpose

Discovery

Discovery

Profiling

Profiling

Screening and validation



Species

Eukaryote

Any

Human, Mouse,Rat

Human

Any

Any

Any

System

Genome Analyzer

Genome Analyzer

iScan System

iScan System

BeadXpress Reader

BeadXpress Reader

BeadXpress Reader

Technology

Sequencing

Sequencing

BeadArray

BeadArray

VeraCode

VeraCode

VeraCode

Page No.

138

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148

155

118

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158

138 www.illumina.com

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mrna-seq mRNA-Seq is a full-length cDNA sequencing appli-

cation that generates a comprehensive, quantitative

view of the entire mRNA portion of the transcrip-

tome. With no probes or primers to design, mRNA-

Seq delivers unbiased sequence information from

any polyA-tailed RNA for analysis of gene expres-

sion, novel transcripts, novel isoforms, alternative

splice sites, allele-specific expression, cSNPs, and

rare transcripts in a single experiment.

Sequencing-based mRNA analysis records the

numerical frequency of a sequence in the library

population, eliminating background signals

observed using relative expression profiles gener-

ated with microarray hybridization technology.

Additionally, data can be recorded and annotated

using current genome information and easily re-

annotated as genome databases evolve.

HIGHLIGHTS

universal discovery platform: Sequence full eukaryotic mRNA without prior sequence information

high sensitivity: Detect single transcripts per cell in as little as 1 µg of total RNA

tunable coverage: Discover rare transcripts with unlimited dynamic range

global validation: Generate genome-wide data comparable to qPCR

sequencing-based gene expressiOn: mrna-seq

Prepare double-stranded cDNA library and ligate adaptersto both ends using standard molecular biology techniques

DAYS 1–2

Amplify cDNA constructs to create clonal clusters of ~1,000copies each on the Cluster Station

DAY 3

Sequence clusters on the Genome Analyzer

DAYS 4–6

mRNA-Seq Workflow

reFerences:

1. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and

quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5: 621–628.

RuN mRNA-SEq ON THE GENOmE ANALYzER

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Technology

Sequencing

1391.800.809.4566 (toll-free in north america) 1.858.202.4566

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PRODuCT SPECIFICATIONS

mRNA-Seq Performance:LImIT OF DETECTION: 1 transcript copy per cell when 1 sample is run per flow cell laneDYNAmIC RANGE: Unlimited and tunable by end userREAD ACCuRACY: > 98.5% with a consensus accuracy of 99.99% at > 3× coverageREPRODuCIBILITY: r2 > 0.99SAmPLE AmOuNT: 1–10 µg total RNA

quantitative Tissue-Specific Expression Detected with mRNA-Seq

the quantity of individual reads are indicated at each genomic location (y-axis). expressed exons are clearly seen as peaks, and are consistent with refseq annotation (bottom). sample-specific expression is quantifiable by comparing results from different samples. the brain sample (top) exhibited 3,115 reads, whereas uHr sample (middle) exhibited 31,109 reads, indicating a ten-fold higher level of expression.

mrna-seq fold-change (brain/uhr)

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r = 0.966

−10 −5 0 5 10

−10

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10

High Concordance between mRNA-Seq and qPCR Results

mrna-seq fold-change results show highly consistent results with qpcr assays for a set of 714 genes.

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sequencing-based gene expressiOn: mrna-seq: reagents

mrna-seq 8-sample prep Kit

mRNA-Seq requires the use of an mRNA-Seq 8-Sample Prep Kit, a Standard Cluster Generation Kit, and a

36-Cycle Illumina Sequencing Kit.

The mRNA-Seq 8-Sample Prep Kit is used to build

DNA libraries for single-read and paired-end

sequencing on the Genome Analyzer. Sample prepa-

ration is straightforward, using standard molecular

biology techniques and requiring minimal hands-on

time. Starting with 1–10 µg total RNA, polyadeny-

lated RNA is purified and fragmented. Unique adapt-

ers that enable attachment to the flow cell surface

are then ligated to each end. Following a short

PCR enrichment, the library is ready to load on the

Cluster Station.mrna-seq 8-sample prep Kit (rs-100-0801)

Isolate poly-A RNASTEP 1

Fragment RNASTEP 2

Make cDNASTEP 3

Ligate adaptersSTEP 4

DAY 1

(1:30)1:30

(0:10)1:00

(0:45)2:00

(0:20)4:30

Total time(hr:min)

(hands-on)(hr:min)

11:30WORKFLOW TOTAL TIME:

Size-Select from gelSTEP 5 (0:30)1:30

Enrich by PCRSTEP 6 (0:30)1:00

(3:45)

mRNA-Seq Assay Workflow

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standard cluster generation Kit v2

The Standard Cluster Generation Kit v2 binds cDNA

libraries prepared using the mRNA-Seq 8-Sample

Prep Kit to the flow cell surface. The Cluster Station

isothermally amplifies the attached cDNA constructs

to create clonal clusters of ~1,000 copies each. Once

reagents are loaded, the fully automated cluster

generation process takes < 4 hours.

standard seq cluster generation Kit (gd-203-2001)

36-cycle illumina sequencing Kit v3

The 36-Cycle Illumina Sequencing Kit v3 contains all

the necessary reagents to directly determine DNA

sequence using sequencing-by-synthesis technology

and the Genome Analyzer. Sequencing-by-synthesis

technology takes advantage of proprietary fluores-

cently labeled, reversibly terminated nucleotides to

sequence, base by base, the millions of clusters gen-

erated on the Cluster Station, in parallel. The ready-

to-load reagents reduce hands-on preparation time

to ten minutes.

36-cycle illumina sequencing Kit v3 (Fc-104-3002)


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sequencing-based gene expressiOn: mrna-seq: reagents


Contents of the mRNA-Seq 8-Sample Prep Kit:

Catalog NumbermRNA-Seq 8-Sample Prep Kit

RS-100-0801

ship at -65° to -85°c. store at -15° to -25°c.

Ultrapure Water 1.8 ml

10 mM Tris Buffer 500 µl

5X Fragmentation Buffer 100 µl

Fragmentation Stop Solution 20 µl

Glycogen 70 µl (20 µg/µl)

Random Primers 15 µl (3 µg/µl)

25 mM dNTPs Mix 200 µl

RNaseOUT Ribonuclease Inhibitor 30 µl (40 units/µl)

GEX Second-Strand Buffer 200 µl

RNaseH 15 µl (2 units/µl)

DNA Polymerase I 50 µl (10 units/µl)

10X End Repair Buffer 500 µl

T4 DNA Polymerase 50 µl (3 units/µl)

Klenow DNA Polymerase 10 µl (5 units/µl)

T4 Polynucleotide Kinase 50 µl (10 units/µl)

10X A-Tailing Buffer 200 µl

1 mM dATP 200 µl

Klenow Exo - 40 µl

2X Rapid T4 DNA Ligase Buffer 1 L

PE Adapter Oligo Mix 100 µl (15 µM)

Rapid T4 DNA Ligase 10 µl (600 units/µl)

5X Phusion Buffer 200 µl

PCR Primer PE 2.0 10 µl (25 µM)

PCR Primer PE 1.0 10 µl (25 µM)

Phusion DNA Polymerase (Finnzymes Oy) 10 µl (2 units/µl)

Bead Binding Buffer 3 ml

Bead Washing Buffer 12 ml

ship at 2° to 8°c. store at 2° to 8°c.

Sera-mag Magnetic Oligo(dT) Beads 0.5 ml

Contents of the Standard Cluster Generation Kit: see page 61.Content of the Illumina Sequencing Kit: see page 56.

ORDERING INFORmATION

RS-100-0801

GD-203-2001

GD-203-2002

FC-104-3002

Contains reagents for preparing eight cDNA libraries.

Contains reagents, one flow cell, one hybridization manifold, and one amplification manifold for preparing clusters on a single flow cell.

Contains reagents, 10 flow cells, 10 hybridization manifolds, and 10 amplification manifolds for preparing clusters on 10 flow cells.

Contains reagents for a 36-cycle run across eight lanes of a single flow cell.

mRNA-Seq 8-Sample Prep Kit (8 libraries)

Standard Cluster Generation Kit v2 (1 flow cell)

Standard Cluster Generation Kit v2 (10 flow cells)

36-Cycle Illumina Sequencing Kit v3 (1 flow cell)


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sequencing-based gene expressiOn: small rna discOvery and analysis

small rna discovery Sequencing-based Small RNA Discovery and

Analysis is a completely open system that enables

small RNA discovery of any size in any organism

without prior sequence or secondary structure

information. With the ability to analyze greater than

four million small RNAs simultaneously in one sam-

ple, Illumina’s Small RNA Discovery and Analysis

System offers the broadest and deepest profiling of

small RNA currently available.

Sequencing-based small RNA discovery records

the numerical frequency of a sequence in the

library population, eliminating background signals

observed using the relative expression profiles from

microarray hybridization technology. Additionally,

data can be recorded and annotated using current

genome information and easily re-annotated as

genome databases evolve.

HIGHLIGHTS

single-day library preparation: Prepare sam-ples in less than one day with only one gel excision

minimal rna input: Start with as little as 1 µg total RNA; does not require mRNA fractionation

universal discovery platform: No prior sequence or secondary structure information required

customiZable siZe selection: Any small RNA between 17–35 nucleotides can be investigatedWide dynamic range: Greater than four million small RNAs can be queried in each flow cell channel

superior data: Robust small RNA identification and quantification

3’ RNA adapter ligation

DAY 1

5’ RNA adapter ligation

Perform RT-PCR amplification

Purify small RNA library

Small RNA Sample Prep v1.5 Workflow

Small RNA Discovery Performance:LImIT OF DETECTION: One transcript copy per cell when one sample is run per flow cell channelDYNAmIC RANGE: Unlimited and tunable by end userSAmPLE AmOuNT: 1 µg total RNA/lane


hsa-let-7b

hsa-let-7f

hsa-let-7a

hsa-MIR-RG-82

has-miR-320

hsa-miR-584

hsa-miR-30a-3p

Small RNA Name Content

Example of Small RNA Discovery and Analysis Data:

76,576

66,100

64,003

1,931

1,647

564

550

RuN SmALL RNA DISCOvERY ON THE GENOmE ANALYzER

illumina scientists analyzed a small rna library isolated from human brain total rna. Over 200 small rna found in the sanger mirna data-base were observed. a portion of the annotated data are shown.

Technology

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sequencing-based gene expressiOn: small rna discOvery and analysis: reagents

small rna sample prep Kit

Sequencing-based Small RNA Discovery and Analysis requires the use of a Small RNA Sample Prep Kit,

Small RNA Cluster Generation Kit, and a 36-Cycle Illumina Sequencing Kit.

Sequencing-based Small RNA Sample Prep Kits

offer researchers the flexibility to customize the

length of the small RNA they wish to investigate,

enabling size-focused or broad size range investi-

gation of different classes of small RNA on one uni-

versal platform. Starting directly from total RNA, the

v1.5 protocol uses modified adaptors to specifically

target microRNAs and other regulatory small RNAs.

small rna sample prep Kit (Fc-102-1009)

Isolate small RNA STEP 1 (1:20)

Start 5’ RNA adapter ligation

STEP 2

Total time (hands-on time):

DAY 1

Complete 3’ RNA adapter ligation

STEP 5

Gel purify small RNA construct library

STEP 7


Complete 5’ RNA adapter ligation

STEP 3

Start 3’ RNA adapter ligation

STEP 4


DAY 2

DAY 3

8:00

(0:10)0:10

(0:30)1:00

(0:10)0:10

(0:30)1:00

(1:00)4:00

(1:30)8:10

(0:40)1:10

(1:20)5:00

WORKFLOW TOTAL TIME: (4:00)15:20

Total time (hands-on time): (0:30)1:00

DAY 4

Perform RT-PCR amplification

STEP 6 1:00 (0:20)

Total time(hr:min)

(hands-on)(hr:min)

Small RNA Sample Prep v1.5 Workflow

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small rna cluster generation Kits

The Illumina Small RNA Cluster Generation Kits

are used to bind total RNA samples prepared with

Small RNA Sample Prep Kits to complementary

adapter oligos grafted on the flow cell surface. The

Cluster Station isothermally amplifies the attached

cDNA constructs to create clonal clusters of roughly

1,000 copies each. Once reagents are loaded, the

fully automated cluster generation process takes

< 4 hours.

small rna cluster generation Kit (Fc-103-1008)

illumina sequencing Kits Illumina Sequencing Kits contain all the reagents

necessary to directly determine the DNA sequence

of each cluster on a flow cell as generated by

the Cluster Station. For Small RNA Discovery

and Analysis, a read length of 36 bp is typically

employed.

36-cycle illumina sequencing Kit v3 (Fc-104-3002)


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PRODuCT SPECIFICATIONS*

Contents of Small RNA Sample Prep Kits:

Catalog Number

Small RNA Sample Prep Reagents

FC-102-1009

Small RNA Oligo Only Sample Prep Kit

FC-102-1013

Ship at -65° to -85°C. Store at -15° to -25°C.

Ultrapure Water 10 ml -

RNaseOUT Ribonuclease Inhibitor 30 µl -

10X Gel Elution Buffer 1 ml -

SRA 0.3 M NaCl 10 ml -

Phusion Polymerase (Finnzymes Oy) 10 µl -

5X Phusion HF Buffer (Finnzymes Oy) 200 µl -

T4 RNA Ligase 30 µl -

10X T4 RNA Ligase Buffer 25 µl -

Primer GX2 10 µl 10 µl

Primer GX1 10 µl 10 µl

SRA RT Primer 10 µl 10 µl

Resuspension Buffer 500 µl -

SRA Gel Loading Dye 350 µl -

25 bp Ladder 45 µl -

25 mM dNTP Mix 10 µl -

Glycogen 70 µl -

SRA Ladder 60 µl -

6X DNA Loading Dye 100 µl -

SRA 5’ Adapter† 15 µl 15 µl

SRA 3’ Adapter 10 µl 10 µl

10X v1.5 sRNA 3’ Adapter 10 µl -

Ship at RT. Store at RT.

SpinX Cellulose Acetate Filters 32 filters -

* Current as of January 2009. Contact Customer Solutions for the most up-to-date information.

† Not required for the v1.5 protocol.

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PRODuCT SPECIFICATIONS (cont.)*

FC-103-1008

FC-103-1009

Contains one flow cell, one amplification manifold, and one hybridization manifold for processing up to eight samples.

Contains ten flow cells, ten amplification manifolds, and ten hybridization manifolds for processing up to 80 samples.

Small RNA Cluster Generation Kit (8 samples)

Small RNA Cluster Generation Kit (80 samples)


FC-102-1009

FC-102-1010

FC-102-1013

Contains reagents for preparing eight total RNA samples (eight samples can be run on one flow cell).

Contains reagents for preparing 40 total RNA samples (40 samples can be run on five flow cells).

Contains only the required RNA adapters (2), RT primer (1), and PCR Primers (2) for preparing 100 samples for small RNA cDNA construct formation. End user must supply all other reagents.

Small RNA Sample Prep Kit (8 samples)

Small RNA Sample Prep Kit (40 samples)

Small RNA Oligo Only Sample Prep Kit (100 samples)

Small RNA Cluster Generation Kits

Small RNA Sample Prep Kits

Illumina Sequencing Kits

FC-104-3002 Contains reagents for generating 36 bp tag sequences across eight lanes of one flow cell.

36-Cycle Illumina Sequencing Kit v3 (1 flow cell)

Contents of the Small RNA Analysis Cluster Kits:

Catalog NumberSmall RNA Analysis Cluster Kit

FC-103-1008

Ship at -65°C to -85°C. Store at -15°C to -25°C.

Cluster Buffer 7.5 ml

Bst DNA Polymerase 380 µl

10 mM dNTPs 750 µl

1M Tris pH 8 65 µl

ddNTPs (130 µM) 35 µl

Terminal Transferase 25 µl

Blocking Buffer 350 µl

Taq DNA Polymerase 30 µl

NlaIII Sequencing Primer 10 µl

Ship at RT. Store at RT.

Hybridization Buffer 12 ml

Wash Buffer 10 ml

Ultrapure Water 50 ml

0.1N NaOH 1.5 ml

Sodium Periodate 68 mg

2N NaOH 20 µl

TE Buffer 1.5 ml

Formamide 35 ml

Storage Buffer 50 ml

Flow Cells 1 per 8 samples

Amplification Manifold 1 per run

Hybridization Manifold 1 per run

Contents of the Illumina Sequencing Kit: see page 56.


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direct HybridizatiOn gene expressiOn

Address Probe

50 bases

Biotin Labeled

cRNA

direct Hybridization assay Illumina Gene Expression Arrays are fully compat-

ible with the standard reagents, techniques, and

tools used extensively throughout the gene expres-

sion community. The protocols feature a first- and

second-strand reverse transcription step, followed

by a single in vitro transcription (IVT) amplification

step that incorporates biotin-labeled nucleotides.

Subsequent steps include array hybridization,

washing, blocking, and streptavadin-Cy3 staining.

Fluorescence emission by Cy3 is quantitatively

detected for downstream analysis. GenomeStudio

software provides results in standard file formats

that can be readily processed with most commercial

expression-analysis software programs.

First-strand synthesisSTEP 1

Second-strand synthesisSTEP 2

cDNA purificationSTEP 3

In vitro transcriptionSTEP 4


DAY 1

cRNA purification and quantitation

STEP 5

Set up hybridizationSTEP 6


DAY 2

Wash and scanSTEP 7


DAY 3

Times are calculated for one technician processing two BeadChips.

(1:15)9:00–19:15

(1:15)17:15–21:15

(3:15)29:00–43:15

= optional stopping pointSTOP

(0:15)2:15

(0:15)STOP

2:15

(0:30)

STOP

0:30

(0:15)STOP

4:00–14:15

(0:45)2:45

(0:45)STOP

2:45

(0:45)16:45–20:45

(0:30)0:30

Total time(hr:min)

(hands-on)(hr:min)

WORKFLOW TOTAL TIME:

HIGHLIGHTS

high reproducibility: 50-mer probes and 100% array QC

high yield: Multiple hybridizations from a single reaction (> 10 µg from just 50 ng of total RNA)

loW sample input requirements: Just 50–100 ng total RNA

loW per-sample cost: Less than half the price of other commercial arrays

Direct Hybridization Assay Workflow

Direct Hybridization Assay Overview

a 50-base gene-specific probe is linked to a short address sequence. this probe is hybridized to labeled nucleic acid derived from total rna.

reFerences:

Kuhn K, Baker S, Chudin E, Lieu M, Oeser S, et al. (2004) A novel, high-performance

random array platform for quantitative gene expression profiling. Genome Research

14: 2347–2356.

RuN DIRECT HYBRIDIzATION GENE EXPRESSION ASSAYS ON THE BEADARRAY READER OR ISCAN SYSTEm

Technology

BeadArray


Gene Expression Assay Performance:DETECTABLE FOLD CHANGE: ≤ 1.35 fold DYNAmIC RANGE: ≥ 3 logsSENSITIvITY: < 1:250,000PRECISION (ARRAY-TO-ARRAY Cv): < 10%

RNA SAmPLE PREPARATION

Options for labeling your RNA sample prior to use with Illumina’s Gene Expression BeadChips, include the TotalPrep and TotalPrep-96 RNA Amplification Kits from Ambion and the TargetAmp Nano-g Biotin-aRNA Labeling Kit from Epicentre. For more information, visit www.illumina.com.

reFerence:

Van Gelder RN, von Xastrow ME, Yool A, Dement DC, Barchas JD, et al.

(1990) Amplified RNA synthesized from limited quantities of heteroge-

neous cDNA. Proc Natl Acad Sci USA 87: 1663–1667.

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direct HybridizatiOn: Whole-genome expression Kits

Whole-genome gene expression Kits

Illumina Whole-Genome Expression Solutions

include complete kits for human, mouse, and rat

whole-genome expression applications. Content for

the Whole-Genome Expression BeadChip is gener-

ated using 50-base, quality-controlled probes and

is subjected to rigorous bioinformatics screening to

ensure optimal sensitivity and specificity.


Whole-genome expression Kit, 6-sample Format (bd-27-302)

Contents of Whole-genome gene expression Kits:

Catalog Number

Whole-Genome Expression Kits (2)

6-, 8-, and 12- sample formats

Human BD-103-0203, BD-101-0203, BD-102-0203

Mouse BD-201-0202, BD-202-0202

Rat BD-27-303

Whole-Genome Expression Kits (6)

6-, 8-, and 12- sample formats

Human BD-103-0603, BD-101-0603, BD-102-0603

Mouse BD-201-0602, BD-202-0602

Rat BD-27-302

ship at rt. store at rt.

Wash E1 BC Buffer 20 ml (4 × 5 ml) 20 ml (4 × 5 ml)

BeadChip Tweezer 1 1

Wash Trays 14 14

Wash Tray Lids 7 7

High-Temperature Wash Buffer 325 ml 325 ml

ship at rt. store at 2° to 8°c.

Blocking E1 Buffer 40 ml 40 ml

Whole-Genome Expression BeadChips 2 6

ship on dry ice. store at -15° to -25°c.

Hybridization E1 Buffer 1.7 ml 1.7 ml

Humidity Control Buffer 2.8 ml 2.8 ml

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y = 0.9537x - 0.3078

r2 = 0.9328

-3 -2 -1 0 1 2 3

2.5

2

1.5

1

0.5

0

-0.5

-1.5

-1

-2.5

-2

fold difference by expression beadchip

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Differential RNA Expression BeadChips vs. qPCR

log-log scatter plot of expression ratios for 20 genes.

Human expression beadchips

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

0 5,000 10,000 15,000 20,000 25,000 30,000

Hum

anH

T-12

HumanWG-6

r2 = 0.993

High concordance between HumanHt-12 v3 and HumanWg-6 v3 beadchip data.

* Illumina guarantees that > 99.99% of the bead types will be present on any given

HumanHT-12 array. This means that up to five HumanWG-6 probes may be repre-

sented with only 0, 1, or 2 copies on each HumanHT-12 array. Using data imputa-

tion from probes surrounding these sites, the GenomeStudio Gene Expression

software module can estimate the presence or absence of the underrepresented

probes in the sample.

The HumanWg-6 beadchip enables the genera-

tion of whole-genome expression profiles for six

samples on a single BeadChip. Each array on the

HumanWG-6 BeadChip provides genome-wide

transcriptional coverage of well-characterized

genes, gene candidates, and splice variants. Probe

content is derived from NCBI RefSeq Release 22

and UniGene Build 188 and specifically designed to

avoid querying pseudogenes and SNP sites.

The HumanHt-12 v3 beadchip is a lower cost,

higher throughput version of the powerful

HumanWG-6 Expression BeadChip, supporting highly

efficient whole-genome expression studies and

expression Quantitative Trait Loci (eQTL) studies.

The BeadChip targets more than 25,000 annotated

genes with more than 48,000 probes per sample*.

High-value content provides genome-wide tran-

scriptional coverage of well-characterized genes,

gene candidates, and splice variants, with a signifi-

cant portion targeting well-established sequences

supported by peer-reviewed literature. A single

HumanHT-12 BeadChip is capable of generating

whole-genome expression profiles for 12 samples.

The Humanref-8 expression beadchip enables

the generation of whole-genome expression

profiles for eight samples on a single BeadChip.

Each array on the HumanRef-8 BeadChip provides

genome-wide transcriptional coverage of well-

characterized National Center for Biotechnology

Information (NCBI) Reference Sequence (RefSeq

Release 22) genes. RefSeq genes are validated,

annotated, and curated on an ongoing basis by

NCBI staff and field experts.


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Kits

BD-101-0203

BD-101-0603

BD-103-0203

BD-103-0603

BD-102-0203

BD-102-0603

BD-20-104

Two HumanWG-6 v3.0 BeadChips. Each BeadChip contains six microarrays allowing for the parallel processing of six samples for greater throughput. Includes two BeadChips, hybridization and wash buffers, and wash trays.

Six HumanWG-6 v3.0 BeadChips. Each BeadChip contains six microarrays allowing for the parallel processing of six samples for greater throughput. Includes six BeadChips, hybridization and wash buffers, and wash trays.

Each HumanHT-12 v3 BeadChip can process 12 samples. Includes two BeadChips plus reagents for hybridization, washing, and processing 24 RNA samples.

Each HumanHT-12 v3 BeadChip can process 12 samples. Includes six BeadChips plus reagents for hybridization, washing, and processing 72 RNA samples.

Two HumanRef-8 v3.0 BeadChips. Each BeadChip contains eight microarrays allowing for the parallel processing of eight samples for greater throughput. Includes two BeadChips, hybridization and wash buffers, and wash trays.

Six HumanRef-8 v3.0 BeadChips. Each BeadChip contains eight microarrays allowing for the parallel processing of eight samples for greater throughput. Includes six BeadChips, hybridization and wash buffers, and wash trays.

Buffer set for use with Expression BeadChips with low-volume seals. Includes sufficient hybridization buffer, wash buffer, and trays to process six BeadChips.

HumanWG-6 v3.0 Expression BeadChip Kit (12 samples)

HumanWG-6 v3.0 Expression BeadChip Kit (36 samples)

HumanHT-12 v3 Expression BeadChip Kit (24 samples)

HumanHT-12 v3 Expression BeadChip Kit (72 samples)

HumanRef-8 v3.0 Expression BeadChip Kit (16 samples)

HumanRef-8 v3.0 Expression BeadChip Kit (48 samples)

multi-Format GEX Buffer Kit


Probe Content Source:

BeadChip

refseq content (build 36.2, release 22)

NM

XM

NR

XR

supplementary content

UniGene (Build 199)

total

HumanHT-12

27,455

7,870

446

196

12,837

48,804


HumanWG-6

27,455

7,870

446

196

12,837

48,804

HumanRef-8

23,811

426

263

26

0

24,526

Contents of Human Expression BeadChip Kits: see page 149.


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mouseWg-6 v2.0 and mouseref-8 v2.0 expression beadchips

-15

-10

10

15

b-cell specific t-cell specific

gene

5

0

-5

log

2 (

b-c

ell

/ t

-ce

ll)

biological validation of mouse-Wg6 v2.0 beadchip performance

messenger rna was purified from cultured mouse b-cell lymphoma line a-20 or t-cell lymphoma line r1.1 using qiagen rneasy columns, amplified, and labeled. (both lines were purchased from american type culture collection.) the labeled products were hybridized to individual arrays (1.5 µg/array) on a mouseWg-6 v2.0 beadchip. log2 ratios of normalized averaged intensity values for the 35 genes examined are shown. bars are coded b-cell specific or t-cell specific based on literature searches performed prior to the experiment. all genes selected by the literature searches are shown, irrespective of array results. in cases where more than one probe existed for a transcript, all probe values were averaged together. thirty-one genes produced ratios corresponding to the literature-based predic-tions; the remaining genes showed no evidence of expression by either cell type.

The MouseWG-6 v2.0 and MouseRef-8 v2.0

Expression BeadChips enable researchers using

the mouse as their model organism to confidently

perform expression profiling experiments. The

BeadChips feature up-to-date content derived from

the National Center for Biotechnology Information

Reference Sequence (NCBI RefSeq) database1

(Build 36, Release 22) supplemented with probes

derived from the Mouse Exonic Evidence Based

Oligonucleotide (MEEBO)2 set as well as exemplar

protein-coding sequences described in the RIKEN

FANTOM23 database.

reFerences:

1. www.ncbi.nlm.nih.gov/RefSeq/

2. www.genome.wustl.edu/activity/ma/products/meebo_mouse_ma.cgi

3. fantom2.gsc.riken.jp


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Kits



BD-201-0202

BD-201-0602

BD-202-0202

BD-202-0602

BD-20-104

Two MouseWG-6 v2.0 BeadChips, each containing > 45,000 probes per array based on RefSeq Release 22 and supplement-ed with MEEBO and RIKEN FANTOM2 content. Each BeadChip contains six microarrays allowing for parallel processing of six samples. Includes hybridization and wash buffers, and wash trays.

Six MouseWG-6 v2.0 BeadChips, each containing > 45,000 probes per array based on RefSeq Release 22 and supplement-ed with MEEBO and RIKEN FANTOM2 content. Each BeadChip contains six microarrays allowing for parallel processing of six samples. Includes hybridization and wash buffers, and wash trays.

Two MouseRef-8 v2.0 BeadChips, each containing > 25,000 probes per array based on RefSeq Release 22 and supplement-ed with MEEBO and RIKEN FANTOM2 content. Each BeadChip contains eight microarrays allowing for parallel processing of eight samples. Includes hybridization and wash buffers, and wash trays.

Six MouseRef-8 v2.0 BeadChips, each containing > 25,000 probes per array based on RefSeq Release 22 and supplement-ed with MEEBO and RIKEN FANTOM2 content. Each BeadChip contains eight microarrays allowing for parallel processing of eight samples. Includes hybridization and wash buffers, and wash trays.

Buffer set for use with Expression BeadChips with low-volume seals. Includes sufficient hybridization buffer, wash buffer, and trays for processing six BeadChips.

mouseWG-6 v2.0 Expression BeadChip Kit (12 samples)

mouseWG-6 v2.0 Expression BeadChip Kit (36 samples)

mouseRef-8 v2.0 Expression BeadChip Kit (16 samples)

mouseRef-8 v2.0 Expression BeadChip Kit (48 samples)


BeadChip mouseWG-6 mouseRef-8


refseq probes (build 36, release 22)

RefSeq NM 26,766 24,854

RefSeq XM 6,856 769

RefSeq NR 56 47

supplementary probes

RIKEN FANTOM2 5,659 0

MEEBO 2,371 0

total 45,281 25,697

Contents of mouse Expression BeadChip Kits: see page 149.


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ratref-12 expression beadchips

The RatRef-12 Expression BeadChip includes con-

tent based largely on the NCBI RefSeq database

(Release 16). Each address and probe sequence has

been carefully selected bioinformatically to cover

major genes of interest. Each BeadChip is capable

of querying 12 samples in parallel, for streamlined

workflow and sample processing.



BD-27-303

BD-27-302

BD-20-104

Two RatRef-12 BeadChips, each containing > 22,000 probes per array targeting all known genes and known alternative splice variants. Each BeadChip contains 12 microarrays, allowing par-allel processing of 12 samples for greater throughput. Includes hybridization and wash buffers, and wash trays.

Six RatRef-12 BeadChips, each containing > 22,000 probes per array targeting all known genes and known alternative splice variants. Each BeadChip contains 12 microarrays, allowing par-allel processing of 12 samples for greater throughput. Includes hybridization and wash buffers, and wash trays.

Buffer set for use with Expression BeadChips with low-volume seals. Includes sufficient hybridization buffer, wash buffer, and trays for six BeadChips.

RatRef-12 Expression BeadChip Kit (24 samples)

RatRef-12 Expression BeadChip Kit (72 samples)



ratref-12 expression beadchip with intelliHyb seal for processing 12 samples simultaneously

reFerences:

For NCBI RefSeq information, visit ftp://ftp.ncbi.nih.gov/refseq/release/

BeadChip

NCBI RefSeq Database (Release 16 NM)

NCBI RefSeq Database (Release 16 XM)

NCBI RefSeq Database (Release 16 XR)

UniGene

total

RatRef-12

6,274

15,983

12

250

22,519

Contents of the RatRef-12 Expression BeadChip Kits: see page 149.


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Query oligo(DAP) annealing, extension,and ligation

Total RNA

PCR with common primersP2

P1P1 P2

b

cDNA synthesisb

Product capturedby hybridizationto BeadChip

P1P1

The Whole-Genome DASL® Assay enables sensitive,

reproducible, cost-effective gene expression pro-

filing of low-abundance and partially degraded

RNA, such as that found in formalin-fixed, paraffin-

embedded (FFPE) samples. Over 24,000 targets can

be analyzed at once. The Whole-Genome DASL

Assay combines the proven DASL (cDNA-mediated

Annealing, Selection, and Ligation) Assay and the

HumanRef-8 Expression BeadChip currently used

in Illumina’s Direct Hybridization Expression Assays

to create an accurate, easy-to-use, higher multiplex

assay. Each BeadChip contains eight arrays for

simultaneous processing of eight samples. Chosen

targets provide genome-wide transcriptional

coverage of well-characterized National Center

for Biotechnology Information (NCBI) Reference

Sequence (RefSeq Build 36.2, Release 22) genes.

HIGHLIGHTS

flexible rna profiling: Work with cell line, fresh-frozen tissue, and FFPE tissue samples

high sensitivity: Start with FFPE tissue, brain tissue, blood samples without globin reduction, or as little as 100 cells

loW-sample input: Use as little as 10–100 ng total RNA from fresh-frozen tissue or 50–200 ng from FFPE tissue

loW per-sample cost: Pay less than one-third the cost of other methods

high-quality results: Obtain high concordance with RT-PCR results (r2 ≈ 0.85)

Whole-genome dasl assay

Humanref-8 expression beadchip

rna prOFiling WitH tHe WHOle-genOme dasl assay

WHOle-genOme dasl assay

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WHOle-genOme dasl assay

Whole-Genome DASL Assay Performance:DYNAmIC RANGE: ~3 logsDETECTABLE FOLD CHANGE: ≥ 1.3–1.5 foldPROBE-LEvEL REPLICATE REPRODuCIBILITY (r2): ≥ 0.99 for intact RNA and ≥ 0.98 for FFPE RNA


Degraded Cell Line RNA

0

10000

20000

30000

40000

50000

60000

70000

0 20000 40000 60000 80000

Raji rep1 raw signal intensity

Raj

i rep

2 ra

w s

igna

l in

tens

ity

y = 0.99x + 89.90

r2 = 0.99

Intact Cell Line RNA

0

10000

20000

30000

40000

50000

60000

0 10000 20000 30000 40000 50000 60000

MCF-7 rep1 raw signal intensity

MC

F-7

rep

2 ra

w s

igna

l in

tens

ity

y = 0.93x + 72.04

r2 = 0.99

FFPE RNA

05000

10000150002000025000300003500040000

0 10000 20000 30000 40000

#98 rep1 raw signal intensity

#98

rep

2 ra

w s

igna

l in

tens

ity

y = 1.09x + 25.17

r2 = 0.99

Reproducible Expression Profiles

Highly reproducible expression profiles (r2 > 0.98) are obtained between technical replicates, using total rna input for cell lines (100 ng, top), artificially degraded samples (200 ng, 95oc for 30 min, middle) and FFpe tissues (200 ng, bottom), respectively.

y = 0.64x - 0.16 r2 = 0.89-4

-2

0

2

4

-4 -2 0 2 4

qRT-PCR: Log2 Raji/MCF-7

WG

-DA

SL: L

og2 F

DR

aji/

MC

F-7

WG-DASL vs. q-PCR

High Concordance Between Whole-Genome DASL Assay and qPCR

High concordance (r2 > 0.85) was obtained between the Wg-dasl assay and qpcr experiments. logarithmic fold-differ-ences (Fd) in transcript abundance in comparisons between two cancer cell lines (raji and mcF-7) were estimated for 24 shared transcripts in both the Wg-dasl assay (fold-difference, y-axis) and qpcr experiments (fold-difference in abundance derived from the cycle threshold, x-axis).

2,500

5,000

7,500

10,000

12,500

15,000

17,500

20,000

frozennat

mea

n n

umb

er o

f pr

obes

det

ecte

d

frozentumor

ffpenat

ffpetumor

0

22,500

14,227

17,111

14,476

17,386

16,329

18,576

15,852

18,292

p < 0.05p < 0.01

mean Number of Detected Probes Across Fresh-Frozen and FFPE Specimens

Four samples were analyzed using the Wg dasl assay. data presented is at either the p < .01 or p < .05 (red) thresholds. error bars indicate observed standard deviations.

Whole-genome dasl assay (cont.)


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Contents of Whole-Genome DASL Assay Kit:

Catalog NumberDA-901-0024DA-901-1024

DA-901-0096DA-901-1096

Number of Samples 24 96

ship -65° to -85°c. store at -15° to -25°c.

cDNA Synthesis Master Mix 700 µl 700 µl

Oligo Hybridization and DNA Binding Buffer 1 3.5 ml 3.5 ml

Master Mix for Extension/Ligation 4.0 ml 4.0 ml

Inoculate PCR Reagent 3.8 ml 3.8 ml

Uracil DNA Glycosylase* 52 µl 52 µl

Single Color Master Mix Reagent 3.2 ml 3.2 ml

XStain BeadChip Solution 4 350 ml 350 ml

Humidity Control Buffer 2.8 ml 5.6 ml (2 x 2.8 ml)

Hybridization Buffer 1.7 ml 1.7 ml

ship -65° to -85°c. store at 2° to 8°c.

Add Master Mix for Extension/Ligation 1 Reagent

15 ml 15 ml

Universal Buffer 1 25 ml 25 ml

Precipitation Solution 1 30 ml 30 ml

Paramagnetic Particles B 2.2 ml 2.2 ml

HumanRef-8 BeadChips 3 12


Make Hyb1 Reagent 7.0 ml (2 x 3.5 ml) 7.0 ml (2 x 3.5 ml)

Wash BeadChip Buffer 20.0 ml (4 x 5.0 ml) 20.0 ml (4 x 5.0 ml)

High-Temperature Wash Buffer 325 ml 325 ml

Universal Buffer 2 300 ml 300 ml

Preparing BeadChip for Hybridization Buffer 950 ml 950 ml


DA-901-0024

DA-901-1024

DA-901-0096

DA-901-1096

Includes one Whole-Genome DASL DAP, three HumanRef-8 BeadChips, each able to analyze eight samples, plus reagents for amplifying, hybridizing, washing, and processing 24 RNA samples.

Includes one Whole-Genome DASL DAP, three HumanRef-8 BeadChips, each able to analyze eight samples, plus reagents for amplifying, hybridizing, washing, and processing 24 RNA samples. Includes UDG enzyme.

Includes one Whole-Genome DASL DAP, twelve HumanRef-8 BeadChips, each able to analyze eight samples, plus reagents for amplifying, hybridizing, washing, and processing 96 RNA samples.

Includes one Whole-Genome DASL DAP, twelve HumanRef-8 BeadChips, each able to analyze eight samples, plus reagents for amplifying, hybridizing, washing, and processing 96 RNA samples. Includes UDG enzyme.

Whole-Genome DASL Assay Kit (24 samples)

Whole-Genome DASL Assay Kit with uDG (24 samples)

Whole-Genome DASL Assay Kit (96 samples)

Whole-Genome DASL Assay Kit with uDG (96 samples)

* Supplied in DA-901-1024 and DA-901-1096 only.


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Query oligo annealing, extension,and ligation

Total RNA

PCR with common primers

Hybridize to the VeraCodeBeadPlate

Address

Address

P1P1

P2P2P3

P1P1

P2P2

P3

b

cDNA synthesisb

Wash the VeraCode BeadPlate

Bind PCR product,elute dye-labeledstrand, prepare for hybridization

Scan the VeraCode BeadPlate

Analyze datausing BeadStudiosoftware

Highabundancetranscript

Lowabundancetranscript

Mediumabundancetranscript

P1P1

P3

veracOde dasl gene expressiOn

veracode dasl assay VeraCode DASL Gene Expression combines the

proven DASL Gene Expression Assay with the

industry-leading data quality of the VeraCode tech-

nology to deliver an extremely robust system for

high-throughput, custom 96- and 384-plex gene

expression analysis. This assay can be used on both

intact and partially degraded samples.

Make activated DNASTEP 1 (0:45)

Add DNA to oligonucleotides, hybridize

STEP 2

Extend, ligate, clean upSTEP 3

Universal PCR cycle at 1,536-plex

STEP 4


DAY 1

STOP

STOP

Wash the VeraCodeBeadPlate

STEP 7

Scan the VeraCodeBeadPlate

STEP 8

Analyze data and generate reports

STEP 9


Bind PCR product, elute dye-labeled strand, prepare for hybridization

STEP 5

Hybridize to the VeraCodeBeadPlate

STEP 6

DAY 2

STOP

STOP

2:00

(0:30)2:30

(1:30)2:00

3:00

(1:00)2:00

(0:10)3:10

(0:10)0:10

1:30

(0:20)0:30

(2:45)9:30

(2:00)7:20

WORKFLOW TOTAL TIME: (4:45)16:50

—

(0:20)

= optional stopping pointSTOP

Total time(hr:min)

(hands-on)(hr:min)

veraCode DASL Assay Workflow

veraCode DASL Assay Overview

the dasl assay monitors gene expression with probe groups to query total rna-generated cdna target sequences.

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HIGHLIGHTS

high sensitivity: Use with intact or partially degraded samples, such as formalin-fixed paraffin-embedded (FFPE) samples

flexible format: Customizable 96- and 384-plex expression analysis

streamlined WorKfloW: Minimize time, volume, and material requirements

RuN THE vERACODE DASL GENE EXPRESSION ASSAY ON THE BEADXPRESS READER.Technology

V

eraCode


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correlations (r2) between replicate assays are greater than 0.99 for fresh-frozen tissues. correlations of replicates from FFpe tissues are nearly as high. in either sample preparation method, as expected, r2 measures are low when comparing two different tissue phe-notypes (normal vs tumor).

103

104

103

104

103

104

103 104 103 104

103

104

103

104

103

104

Normal vs Normal Normal vs Tumor Tumor vs Tumor

Normal vs Normal Normal vs Tumor Tumor vs Tumor

103 104

r2 = 0.983 r2 = 0.698 r2 = 0.994

r2 = 0.992 r2 = 0.724 r2 = 0.993

103 104 103 104 103 104

Normal Prostate A

Normal Prostate A

Nor

mal

Pro

stat

e B

Nor

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Pro

stat

e B

Tumor Prostate A

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B

Tumor Prostate

Nor

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Pro

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Tumor Prostate ATu

mor

Pro

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e B

Tumor Prostate

Nor

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Pro

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e

a: Fresh-Frozen prostate tissue

b: Formalin-Fixed paraffin-embedded prostate tissue


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veracOde dasl gene expressiOn: reagents

veracode dasl gene expression analysis Kits

The VeraCode DASL Gene Expression Analysis

Kits include DNA activation reagents, DASL assay

reagents, and VeraCode BeadPlates.

Simply choose your input:- Gene List- Region(s) of Interest- RS List- Sequence

Preliminary DAP Design File

Evaluation

Final DAP File

Submit order via eCommerce or

Technical Support

Illumina Assay Design Tool

1–2 business daysfrom submission

to reportAdd or modify as many times as you like

Custom Pool DASL Assay Panel (DAP) Design and Ordering Process

Knowledgeable illumina scientists support the efficient custom design ordering process.

veracode dasl gene expression analysis Kit (vc-201-2096)

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vC-201-2096

vC-201-2384

The kit contains sufficient reagents to run 480 samples and includes a custom 96-plex DAP, five VeraCode 96-plex Bead Plates, cDNA synthesis reagents, and DASL gene expression analysis reagents for VeraCode.

The kit contains sufficient reagents to run 480 samples and includes a custom 384-plex DAP, five VeraCode 384-plex Bead Plates, cDNA synthesis reagents, and DASL gene expression analysis reagents for VeraCode.

veraCode DASL Gene Expression Analysis Kit (96-plex, 480 samples)

veraCode DASL Gene Expression Analysis Kit (384-plex, 480 samples)

Contents of veraCode DASL Gene Expression Kit:

Catalog NumberveraCode DASL Gene Expression Kit

VC-201-2096


Master Mix cDNA Synthesis for Single Use Reagent 700 µl

Oligo Hybridization and DNA Binding Buffer 1 17.5 ml (3 × 3.5 ml)

Master Mix for PCR Reagent 16 ml (3 × 3.2 ml)

Add Master Mix for Extension and Ligation 1 Reagent 100 ml

ship at -65° to -85°c. store at 4°c.

Inoculate PCR Reagent 19 ml (3 × 3.8 ml)

Universal Buffer 1 250 ml

Master Mix for Extension and Ligation 20 ml (5 × 4 ml)

ship at rt. store at 2° to 8°c.

Magnetic Particle B Reagent 11 ml (5 × 2.2 ml)

Make Hybridization 2 Buffer 35 ml (10 × 3.5 ml)

VeraCode BeadPlates (96- or 384-plex) 5 each

ship at rt. store at rt.

Universal Wash Buffer 2 300 ml

VeraCode Wash Buffer 250 ml

ship at rt. store at -15° to -25°c.

VeraCode DAP (custom 96- or 384-plex DASL assay pool) 6 ml (5 × 1.2 ml)

Gene Expression Analysis - Illumina, Inc. · GENE EXPRESSION ANALYSIS sequencing-based gene...

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