**MAQC Main Study -- Illumina Experimental Plan**

# Test Sites

## (1) Illumina

> Shawn C. Baker
>
> 9885 Towne Centre Dr.
>
> San Diego, CA 92121
>
> 858-202-4597

## (2) University of Texas Southwestern Medical Center

> Quan Li, Ph.D.
>
> Assistant Professor
>
> Microarray Core Facility
>
> UTSW Medical Center
>
> 6000 Harry Hines Blvd./ND6.504
>
> Dallas TX 75390-8814

## (3)Burnham Institute

> Craig Hauser
>
> Cancer Center Associate Director, Shared Resources;
>
> Director of Research Support
>
> The Burnham Institute
>
> 10901 North Torrey Pines Rd.
>
> La Jolla, CA 92037

# Required Materials

## Equipment

> Safety glasses
>
> Lab coats
>
> Single-channel precision pipet, p2, p20 and p100
>
> Tube vortexer
>
> Table-top centrifuge with 96-well plate carriers (for drying slides)
>
> Microcentrifuge (strip tube compatible)
>
> Vacuum centrifuge concentrator (strip tube compatible)
>
> NanoDrop ND-1000A UV-Vis Spectrophotometer (or equivalent device for
> measuring RNA OD260/280)
>
> Agilent 2100 Bioanalyzer
>
> Thermal cycler with adjustable-temperature heated
> lid--or--hybridization ovens set at 42ºC, 37ºC and 16ºC heat block
>
> Orbital shaker
>
> Rocker mixer
>
> Scigene Hybex^TM^ Microarray incubation system with waterbath insert
>
> Compressed air source (at least 50 psi) for isolation table
>
> Software: BeadScan 2.3.0.10 and BeadStudio 1.5.1.3

## Disposables

> Clean paper towels
>
> RNAse-free 0.5 ml microfuge tubes (Note: we recommend the use of strip
> tubes when you are labeling multiple samples.)
>
> RNAse-free 15 ml disposable conical tubes (for blocking and SA-Cy3
> steps)
>
> Powder-free protective gloves

## Kits and Reagents

> Illumina RNA Amplification Kit (Ambion, Inc., catalog \# I1755) --
> **supplied by Illumina**
>
> Biotin-16-UTP, 10mM (Perkin Elmer) -- **supplied by Illumina**
>
> Illumina High-temp wash buffer (Illumina part number 11206039) --
> **supplied by Illumina**
>
> Four (4) Sentrix Human-6 Expression BeadChips, 48k v1.0 (Illumina,
> catalog# BD-25-101) -- **supplied by Illumina**
>
> Human-6 buffer kit (Illumina, included with chips) -- **supplied by
> Illumina**
>
> Control RNA sample -- **supplied by Illumina**
>
> Ambion's Human Brain Reference RNA (HBRR) -- to be delivered by Ambion
>
> Stratagene's Universal Human Reference RNA (SUHRR, Catalog number:
> 740000) -- **to be delivered by Ambion**
>
> Mixture #1 (75% HBRR/25% SUHRR) -- **to be delivered by Ambion**
>
> Mixture #2 (25% HBRR/75% SUHRR) -- **to be delivered by Ambion**
>
> 100% ethanol
>
> Deionized formamide (Fischer Scientific, catalog \# 4610)
>
> Streptavidin-Cy3 ( FluoroLink™ Cy™3, Amersham Biosciences catalog \#
> PA43001, diluted to 1mg/mL or 1 µg/µL with RNase-free water ); NOTE:
> Once the SA-Cy3 has been reconstituted, Illumina recommends making 20
> µL aliquots. Store these aliquots at -20°C. Once the aliquots have
> been thawed for use, store them in the dark at 4°C for up to one
> month. DO NOT FREEZE AND THAW REPEATEDLY.
>
> RNase-free water

# Experimental Design

> Five (5) replicate sample labelings and hybridizations will be
> performed on the following four (4) tissue mixtures:
>
> SUHRR (A)
>
> HBRR (B)
>
> 25% HBRR/75% SUHRR (C)
>
> 75% HBRR/25% SUHRR (D)

# Sample Preparation

> All samples should be processed at the same time by the same person.

## Total RNA QC

> The SUHRR sample will be delivered in 70% ethanol and 0.1M sodium
> acetate and should be prepared according to Stratagene's recommended
> procedure. The HBRR and mixture samples will be delivered as aqueous
> solutions. For Site 2, all samples will be delivered by Illumina as
> aqueous solutions. Before proceeding to the sample labeling step, all
> total RNA samples should be quantitated with a NanoDrop
> spectrophotometer (or equivalent) and the OD260 and OD280 measurements
> should be recorded and reported to the MAQC study. (See "MAQC Sample
> Processing Overview" document for details.)
>
> A quality assessment should be made by running 200ng of each of the
> four samples on the Bioanalyzer system. The Bioanalyzer traces should
> be saved along with the 28S/18S ratios and RIN values. (See "MAQC
> Sample Processing Overview" document for details.)

## Sample Labeling

> All 20 sample labeling reactions should be performed in parallel by
> the same operator. For each replicate, 200ng total RNA should be
> labeled according to the procedure described in the manual included in
> the "Illumina® RNA Amplification Kit". The IVT reaction should be run
> for 16 hours.

## Labeled cRNA QC

> Before proceeding to the sample hybridization step, all cRNA samples
> should be quantitated with a NanoDrop spectrophotometer (or
> equivalent) and the OD260 and OD280 measurements should be recorded
> and reported to the MAQC study. (See "MAQC Sample Processing Overview"
> document for details)
>
> The fragment size distributions should be made by running 200ng of
> each of the 20 replicates on the Bioanalyzer system using the
> Eukaryotic mRNA Assay with smear analysis. The Bioanalyzer traces
> should be saved. (See "MAQC Sample Processing Overview" document for
> details.)

# Sample Hybridization

> Samples should be prepared for hybridization according to the "Gene
> Expression on Sentrix® Arrays Direct Hybridization System Manual" with
> the addition of a ten-minute wash in Illumina High-temp wash buffer at
> 55°C for 10 minutes in a Scigene Hybex Microarray Incubation System
> with a waterbath insert following the overnight hybridization. All
> hybridizations should be performed in parallel by the same operator.
>
> 1.5ug cRNA should be used for each hybridization.
>
> The following sample layout should be used:
>
> ![](media/image1.wmf){width="3.345138888888889in"
> height="1.3868055555555556in"}
>
> "Control" is an Illumina control sample to be used only for internal
> purposes (it is not part of the MAQC study and those samples should
> not be reported).
>
> Hybridizations should be carried out for 16 hours at 55oC.
>
> All four slides should be washed and processed in parallel.

# Scanning

> The chips should be scanned as soon as possible after the final dry
> down procedure using BeadScan 2.3.0.10 at a multiplier setting of two
> (2).
>
> The slides should be scanned in order with chips 1, 2 and 3 batched
> together in the first scanner run and chip 4 in the second scanner
> run.

# Data Analysis/Outputs

> The microarray images will be registered and extracted automatically
> during the scan according to the manufacturer's default settings.
>
> BeadStudio version 1.5.1.3 should be used. The samples should be named
> according to the standard MAQC convention (e.g., A1, A2, A3, A4, A5,
> B1, etc.) and generated twice in two rounds of processing. The first
> processing should use no normalization. The second should use the
> cubic spline normalization method. The reference group for the
> normalized experiment should be a group containing all of the brain
> samples (B1, B2, B3, and B4). Because this group is only used for the
> sake of normalization, all columns pertaining to the group should be
> deleted from the gene_profile file prior to data submission. For each
> experiment, each site should generate standard text output containing
> the following fields:

-   AVG_Signal

-   BEAD_STDEV

-   Avg_NBEADS

-   Detection

> The naming conventions for the files shall be as follows:

  --------------- --------------------------------- --------------------------------
  Site            Normalized file name              Raw data file name

  Illumina        ILM_1_ALL_norm_gene_profile.csv   ILM_1_ALL_raw_gene_profile.csv

  UT Southwestern ILM_2_ALL_norm_gene_profile.csv   ILM_2_ALL_raw_gene_profile.csv

  Burnham         ILM_3_ALL_norm_gene_profile.csv   ILM_3_ALL_raw_gene_profile.csv
  Institute                                         
  --------------- --------------------------------- --------------------------------

> For each site, these files plus the additional files generated by the
> scanner (.tif, .IDAT, .locs, .sdf, .csv and .xml) will be zipped into
> a Winzip compressed file and submitted to the MAQC FTP site per
> instructions provided by Leming Shi.
>
> In addition, the normalized file shall be processed outside of the
> BeadStudio software to eliminate negative hybridization signal values
> by adding an offset constant. This constant will be chosen such that
> the lowest AVG_Signal value for each sample has a value of 1. The
> processing is necessary because some of the analyses to be conducted
> by the MAQC group will not work with negative values. Because this
> processing occurs outside of the normal BeadStudio software, it will
> be conducted by Illumina using data provided by the third-party sites.