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Special for MAQC Test site use

Hybridization Protocol for CustomArray™12K Chips

&

Preparation for Scanning

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# CustomArray assay workflow 

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Hybridization and Imaging Protocol

# Table of Contents {#table-of-contents .TOC-Title}

#  {#section .TOC-Title}

[**CustomArray assay workflow i**](#customarray-assay-workflow)

[Table of Contents ii](#table-of-contents)

[Introduction 1](#introduction)

[Materials provided 1](#materials-provided)

[Materials required but not supplied
1](#materials-required-but-not-supplied)

[Assemble chips with hybridization caps, gaskets and clamps
2](#assemble-chips-with-hybridization-caps-gaskets-and-clamps)

[Preparation of solutions 3](#preparation-of-solutions)

[(MAQC Test Sites will be provided with a complete buffer kit)
3](#__RefHeading___Toc112140021)

[Solutions common for all types of samples (biotinylated and
fluorochrome incorporated)
3](#solutions-common-for-all-types-of-samples-biotinylated-and-fluorochrome-incorporated)

[Solutions required only for biotinylated samples
5](#solutions-required-only-for-biotinylated-samples)

[Pre-Hybridization 5](#pre-hybridization)

[RNA Fragmentation (RNA target samples only)(provided)
6](#rna-fragmentation-rna-target-samples-onlyprovided)

[Hybridization 6](#hybridization)

[Hybridization Washing 7](#hybridization-washing)

[Post-hybridization labeling and washing for biotinylated samples
7](#post-hybridization-labeling-and-washing-for-biotinylated-samples)

[Final Washing 8](#final-washing)

[Preparing the CustomArray12K Chip for Scanning
8](#preparing-the-customarray12k-chip-for-scanning)

Hybridization and Imaging Protocol

# Introduction

This guide explains how to hybridize a labeled target sample (DNA or
RNA) to the CombiMatrix CustomArray^TM^12K chip, and prepare the array
for scanning. Preparing the labeled target samples is the user's
responsibility. We recommend usage of 5 μg of a labeled target sample
per one array. If two labeled samples are hybridized to the same array
(a two-color hybridization scheme), use 5 μg of each sample (totally 10
μg). This protocol is optimized for gene expression analysis. Other
applications, such as comparative genome hybridization and genome
tiling, may require the user to optimize hybridization conditions. Both
biotin-streptavidin detection system (biotin-labeled nucleic acid
targets and streptavidin-conjugated fluorochrome) and
fluorochrome-labeled nucleic acid targets could be used with
CustomArray^TM^12K chips. Arrays are scanned with a high-resolution
fluorescent microarray scanner that is compatible with a 1"x3" slide
format and has a minimum resolution of 5μm and adjustable focus.

## Materials provided

CustomArray^TM^ 12K chip

> IMPORTANT! **Do not touch the chip surface. Keep in a dry cool place.
> Store up to 4 months.**

Hybridization cap

Gasket (O-ring)

Clamps

LifterSlip^TM^ coverslip for imaging

Imaging Solution

## 

## Materials required but not supplied

Labeled target (DNA or RNA) samples.

Pipettors and tips

Sterile plastic ware

Gloves (powder-free)

Adhesive tape (Scotch tape)

Water (RNase and DNase-free)

50x Denhardt's solution

EDTA

Tween-20

20x SSPE buffer

10 mg/ml sonicated salmon sperm DNA

1% SDS

Deionized (DI) Formamide (if working with labeled RNA)

Phosphate buffered saline (PBS)

Acetylated Bovine Serum Albumin (BSA)

Reagents for RNA fragmentation (if working with labeled RNA)

Reagents for biotin-streptavidin detection system (if working with
biotinylated samples)

Rotisserie oven or rotating incubator for array hybridization

CustomArray holders for rotisserie oven (rotating incubator)

Standard, high-resolution fluorescent microarray scanner (CombiMatrix
recommends the Axon Instruments Genepix® 4000B and 4200A microarray
scanners, Applied Precision arrayWoRx® Biochip Reader, and the Perkin
Elmer ScanArray® microarray scanner).

# Assemble chips with hybridization caps, gaskets and clamps 

IMPORTANT! Hybridization Cap, Gasket, and Clamps are intended for single
use only. Dispose of these items upon completion of this protocol.

1.  Place the gasket into groove on hybridization cap.

2.  Position the hybridization cap over the slide so that the bottom
    edge of the slide is resting against the stop on the hybridization
    cap and centered over slide.

3.  Secure the hybridization cap in place with clamps provided (see
    Figure 1).

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**Figure 1** CustomArray^TM^12K and accessory components.

NOTE: Throughout this protocol, use a 200 μl micropipettor with
RNase-free pipet tip to add or remove solutions through the
hybridization cap portals. Hold the chip with the assembled
hybridization cap at a 45 degree angle (as shown on the cover
photograph) and add/remove solutions through the lower portal.

IMPORTANT! Do not allow the array to become dry at any step in the
protocol. Proceed rapidly when changing solutions during the
pre-hybridization, hybridization, and washing steps. Do not leave the
hybridization chamber empty for any significant length of time (have it
filled).

# Preparation of solutions

#  (MAQC Test Sites will be provided with a complete buffer kit)

IMPORTANT! The solution set is different for biotinylated and
fluorochrome incorporated target samples. Usage of biotinylated samples
requires additional post-hybridization labeling and extra washing steps
(see the Assay workflow). Thus, the user will need three additional
solutions (not required for fluorochrome-incorporated samples).

NOTE: The hybridization buffer composition depends on the type of
nucleic acids used as labeled targets. For RNA (but not DNA) targets, it
should include 25% formamide.

## Solutions common for all types of samples (biotinylated and fluorochrome incorporated)

1\. Prepare the 2X Hyb Solution Stock (see Table). This stock will be
used for preparation of the Pre-hybridization and Hybridization
Solutions. All reagents should be RNase and DNase-free. The prepared 2x
Hyb Solution Stock should be filter-sterilized and stored at room
temperature.

  ------------------------------- ------------ ---------------------------
  2X Hyb Solution Stock (all                   
  types of samples) (Provided)                 

  Reagent                         Volume for   Final Concentration
                                  10 ml        

  20X SSPE                        6 ml         12X

  10% Tween-20                    100 μl       0.1%

  0.5M EDTA                       560 μl       40mM^a^

  RNase-free water                to 10 ml     

  Total Volume                    10 ml        
  ------------------------------- ------------ ---------------------------

> ^a^Final concentration includes the EDTA from the SSPE.

2\. Prepare the Pre-hybridization Solution, 100 μl per one array (see
Table).

^a^Salmon sperm DNA should be heated to 95°C for at least 5 min and then
placed on ice for at least 1 minute before use.

This solution should be prepared fresh each time you hybridize arrays.

3\. Prepare the Hybridization Solution (see Table). We recommend using
approximately 5 μg of labeled DNA or RNA per array, thus, the initial
concentration of a labeled sample prior to addition to the Hybridization
Solution should be at least 0.25 μg/μl. For best results, labeled RNA
should be fragmented to 50-200 base length prior to adding to the
Hybridization Solution (see the section "RNA Fragmentation").

  ------------------------- ----------------- ---------------------------
  Hybridization Solution                      
  (all types of samples)                      

  Reagent                   Volume per        Final Concentration
                            CustomArray12K    
                            Chip (μl)         

  2X Blocking/Hybridization 50                6X SSPE, 0.05% Tween-20,
  solution                                    20mM EDTA

  DI Formamide (RNA         25                25%
  targets)                                    

  fragmented RNA            5ug(up to 20 μl)  50 ng/μl recommended

  Salmon sperm DNA          1                 100 ng/μl
  (10mg/ml)                                   

  1% SDS                    4                 0.05%

  RNase-free water          to 100            

  Total Volume              100 μl            
  ------------------------- ----------------- ---------------------------

NOTE : Replace formamide with water when working with the labeled DNA
targets.

4\. Prepare the Hybridization Wash (HW) Solutions (see Table). The
prepared HW Solutions should be filter-sterilized and stored at room
temperature.

  -------------------------------- ------------------- ------------------
  Hybridization Wash Solutions &                       
  Incubation Times (all types of                       
  samples)                                             

  Solution                         Temperature (°C)    Incubation Time
                                                       (min)

  Wash 1 (HW1): 6x SSPE, 0.05%     Pre-heat to         5
  Tween-20                         hybridization       
                                   temperature         

  Wash 2 (HW2): 3x SSPE, 0.05%     RT                  5
  Tween-20                                             

  Wash 3 (HW3): 0.5x SSPE, 0.05%   RT                  5
  Tween-20                                             

  Wash 4 (HW4): 2x PBS, 0.1%       RT                  5
  Tween-20                                             
  -------------------------------- ------------------- ------------------

5\. Prepare the Final Wash (FW) Solution (see Table). The prepared FW
Solution should be filter-sterilized and stored at room temperature.

  ------------------------- ---------------------- -----------------------
  Final Wash (all types of                         
  samples)                                         

  Solution                  Temperature (°C)       Incubation Time (min)

  Final Wash (FW): 2X PBS   RT                     Twice 5
  ------------------------- ---------------------- -----------------------

## 

## Solutions required only for biotinylated samples

If working with fluorochrome-labeled targets, skip this step. If working
with biotinylated targets, prepare additional Post-hybridization
Blocking (BB), Labeling (BL) and Wash (BW) Solutions (see Table). The
prepared BB and BW Solutions should be filter-sterilized and stored at
room temperature. BL Solution should be prepared fresh each time you
hybridize arrays.

  ----------------------------------------- -------------- --------------
  Post-hybridization Blocking, Labeling and                
  Wash Solutions (biotinylated samples)                    

  Solution                                  Temperature    Incubation
                                            (°C)           Time (min)

  Biotin Blocking solution (BB): 2X PBS,    RT             15
  0.1% Tween-20, 1% BSA                                    

  Biotin Labeling solution (BL): 1 μg/ml    RT             30
  streptavidin-conjugated fluorochrome, 2X                 
  PBS, 0.1% Tween-20, 1% BSA                               

  Biotin Wash (BW): 2X PBS, 0.1% Tween-20   RT             Twice 5
  ----------------------------------------- -------------- --------------

NOTE: To prepare the Biotin Labeling Solution (BL), dilute
streptavidin-conjugated fluorochrome with the Biotin Blocking Solution
(BB). For example, if you use Fluorolink^TM^ Cy5-labeled streptavidin
from Amersham (cat \# PA45001, 1 mg of dry powder), dissolve it to 1
mg/ml stock, store as aliquots in a freezer, thaw an aliquot and dilute
1:1000 in the BB Solution (1 μl per 1 ml of BB, discard the used
aliquot).

# Pre-Hybridization

NOTE : The Combimatrix CustomArray 12K chips are supplied dry, they need
to be re-hydrated prior to hybridization, and then pre-hybridized to
block non-specific binding of nucleic acid targets.

1.  Thaw provided aliquot of pre hybridization buffer @ 37oC

2.  Place Aliquot of Salmon Sperm at 95oC for 5min place on Ice for 1
    min

3.  Add 10ul of Salmon Sperm to the Pre Hybridization solution, place on
    ice

4.  Pre-heat an incubator to 65^o^C. Set a hybridization (rotisserie)
    oven to 45^o^C hybridization temperature. See Table for suggestions
    on hybridization temperatures for different sample types and
    buffers. These temperatures are recommended for standard
    oligonucleotide lengths (35-40mers) and have been optimized for gene
    expression applications, thus, they should be used only as a guide
    and may be optimized empirically.

  --------------- ------------------------------------- ----------------------
  Hybridization                                         
  Temperature                                           

  Sample          Hybridization Solution                Hybridization
                                                        Temperature (°C)

  RNA             Hybe solution + 25% Formamide         45

                                                        
  --------------- ------------------------------------- ----------------------

5.  Fill hybridization chamber with room temperature RNase- and
    DNase-free water (approximately 100 ul). Make sure not to introduce
    air bubbles. Cover the hybridization cap portals with non-permeable
    adhesive tape (for example, Scotch tape) to prevent evaporation and
    air bubble formation.

6.  Incubate at 65^o^C for 10 min in the pre-heated incubator.

7.  Remove array from the incubator and bring to room temperature.

8.  Remove the tape and pipet water out of hybridization chamber.

9.  Fill hybridization chamber with the prepared Pre-hybridization
    Solution. Mix gently by pipetting. Make sure not to introduce air
    bubbles. Cover the hybridization cap portals with non-permeable
    adhesive tape.

10. Incubate at 45^o^C hybridization temperature for 30 min in the
    pre-heated hybridization oven.

# RNA Fragmentation (RNA target samples only)(provided)

NOTE: Labeled RNA target samples should be fragmented to maximize
binding specificity and detection sensitivity. We have found that 50 to
200-base fragments work best. You could use either the protocol
described below, or a protocol that is established in your laboratory.

1.  Prepare the 5x RNA Fragmentation Solution (see Table). Filter
    sterilize the prepared solution and store at room temperature

  ------------------------- ----------------- ---------------------------
  5x RNA Fragmentation                        
  Solution (RNA samples                       
  only)                                       

  Reagent                   Volume for 10 ml  Final Concentration

  1 M Tris Acetate pH 8.1   2 ml              200 mM
  (adjust pH with glacial                     
  acetic acid)                                

  KOAc                      0.49 g            500 mM

  MgOAc                     0.32 g            150 mM

  Water                     To 10 ml          

  Total volume              10 ml^a^          
  ------------------------- ----------------- ---------------------------

2.  Take 5 μg of the labeled RNA dissolved in 16 μl of water (thus, the
    initial concentration of labeled RNA samples should exceed 0.3
    μg/μl). Add 4ul of 5x RNA Fragmentation Solution.

3.  Incubate at 95^o^C for 20 min. Place on ice.

4.  Use the whole volume (20 μl) to add to the Hybridization Solution.

# Hybridization

1.  Denature the Hybridization Solution at 95^o^C for three minutes, and
    then cool briefly on ice.

2.  Spin at maximum speed in a microcentrifuge for one minute.

3.  Remove Pre-hybridization Solution from the hybridization chamber.

4.  Add 100 μl of Hybridization Solution and mix gently with repeated
    pipetting. Make sure not to introduce air bubbles if the arrays are
    incubated without rotation. A small air bubble can be introduced to
    improve the mixing process if the arrays are rotated in the
    hybridization rotisserie oven.

5.  Carefully wipe excess solution from the surface of the hybridization
    cap with a lint-free tissue and cover the hybridization cap portals
    with non-permeable adhesive tape.

6.  Incubate at the 45^o^C hybridization temperature for 16 hours in the
    hybridization rotisserie oven.

NOTE: To improve array performance, usage of a rotisserie oven or a
rotating incubator is recommended to ensure mixing during hybridization.
You can introduce a small air bubble into the hybridization chamber to
improve the mixing process during rotation. CustomArray chips can be
attached to standard rotisseries using various models of holders
manufactured by Combimatrix.

# Hybridization Washing

IMPORTANT! Do not allow the array to become dry at any step in the
protocol. Proceed rapidly when changing solutions during the washing
steps. Do not leave the hybridization chamber empty for any significant
length of time.

NOTE 1: Keep the hybridization cap in place during all wash steps. Hold
the chip with the assembled hybridization cap at a 45 degree angle (as
shown on the cover photograph) and add/remove solutions through the
lower portal by using a 200 μl micropipette with RNase-free pipet tip.

NOTE 2: For every wash step, we recommend to rinse the hybridization
chamber with the corresponding Wash Solution prior to the wash
incubation. Add Wash Solution into the chamber, gently mix by pipetting,
remove it, and fill the chamber again with the same solution.

NOTE 3: Except for the first wash step, all subsequent wash incubations
could be reduced to 1 min. However, if processing multiple arrays, you
could extend the wash incubation time until you rinse and fill all
hybridization chambers.

NOTE 4: Keep the chips protected from light during all long (more than 5
min) incubations (cover with foil).

1.  Prior to starting the wash procedure, preheat the HW1 Solution to
    the hybridization temperature.

2.  Remove the Hybridization Solution and rinse the hybridization
    chamber with HW1 Solution.

3.  Add HW1 Solution to the hybridization chamber and incubate the
    hybridization chamber at the hybridization temperature for 5
    minutes.

4.  Remove HW1 Solution and rinse the hybridization chamber with HW2
    Solution.

5.  Add HW2 Solution to the hybridization chamber, mix gently by pipet,
    and incubate at room temperature for 1-5 minutes (see NOTE 3).

6.  Remove HW2 Solution and rinse the hybridization chamber with HW3
    Solution.

7.  Add HW3 Solution to the hybridization chamber, mix gently by pipet,
    and incubate at room temperature for 1-5 minutes (see NOTE 3).

8.  Remove HW3 Solution and rinse the hybridization chamber with HW4
    Solution.

9.  Add HW4 Solution to the hybridization chamber and mix gently by
    pipet.

    **Biotinylated samples**: Retain HW4 Solution in the hybridization
    chamber until you are ready to perform the blocking and labeling
    procedures.

**Fluorochrome-labeled** **samples:** Proceed to the final wash.

#  Post-hybridization labeling and washing for biotinylated samples

1\. Remove HW4 Solution from the hybridization chamber.

2\. Add Biotin Blocking (BB) Solution to the hybridization chamber and
gently mix by pipet.

3\. Incubate at room temperature for 15 minutes (protect from light),
and then remove the BB Solution.

> 4\. Add the Biotin Labeling (BL) Solution to the hybridization
> chamber, gently mix by pipet, and cover the hybridization cap portals
> with non-permeable adhesive tape.

5\. Incubate at room temperature for 30 minutes (protect from light).

6\. Remove BL Solution from the hybridization chamber.

7\. Rinse the hybridization chamber with the Biotin Wash (BW) Solution.

8\. Add BW Solution, mix gently by pipet, and incubate at room
temperature for 1-5 min.

9\. Repeat washing with BW Solution one more time.

# Final Washing

1.  Remove the HW4 (fluorochrome-labeled samples), or BW (biotin-labeled
    samples) Solutions from the hybridization chamber.

2.  Rinse the hybridization chamber with the final wash (FW) solution.

3.  Add the FW solution and incubate for 1-5 minutes.

4.  Repeat washing with FW solution one more time. Keep array
    hybridization chambers filled with FW solution until you are ready
    to prepare them for scanning.

# Preparing the CustomArray12K Chip for Scanning

The CustomArray chip must be scanned wet using the Imaging Solution
supplied. The LifterSlip™ coverslip (supplied) retains buffer on the
chip without damaging the chip surface. Due to its specially designed
edges, the LifterSlip™ coverslip does not contact the chip surface.

IMPORTANT! Do not use a standard coverslip with the CustomArray chip.
Only use the LifterSlip™ coverslip, otherwise the chip may be damaged.

> ![](media/image8.png){width="4.0055555555555555in"
> height="3.0993055555555555in"}
>
> **Figure 2** CustomArray™12K with LifterSlip™ coverslip.

1.  Remove the FW solution from the hybridization chamber.

2.  Add Imaging Solution (supplied with arrays) to the hybridization
    chamber.

3.  Carefully remove the hybridization chamber from the array by
    removing clamps and lifting the hybridization cap off the slide
    surface, being careful to retain Imaging Solution on the surface of
    the array (be ready to add more using a micropipette if array is not
    completely covered).

4.  Use thin-tipped forceps to hold the LifterSlip™ coverslip so that
    the raised edges face the slide (the side with edges could be
    detected by rubbing with forceps, the edges feel rough).

5.  Lay the LifterSlip™ coverslip at an angle onto the slide so that it
    is centered over the array (see Figure 2). First touch the imaging
    solution covering the array with one side of the LifterSlip, then
    slowly lower it down, taking care not to introduce air bubbles. If
    the bubbles still form, lift one side of the LifterSlip with forceps
    (or a razor blade) to let the bubbles out, and lower it down again.

6.  Carefully remove any excess Imaging Solution from the edge of the
    LifterSlip using a lint-free tissue until the it is resting on its
    raised edges.

7.  Load the CustomArray chip into the scanner taking care not to
    disturb the LifterSlip™ coverslip.