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cDNA microarray experiment: set up and execution
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cDNA microarray experiment: set up and execution

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Category
Glycosyltransferases & related proteins
Protocol Name

cDNA microarray experiment: set up and execution

Authors
Takematsu, Hiromu *
Laboratory of Biochemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University

Fujinawa, Reiko
-, RIKEN ASI
*To whom correspondence should be addressed.
KeyWords
Reagents

RNA purification

  • mRNA isolation kit (mTRAP mRNA isolation kit, Active Motif Cat#23024)
  • DNase I RNase free (2U/uL Applied Biosystems/Life Technologies, Carlsbad, CA, Cat#AM2222)
  • Disposable syringe 1mL
  • 18–21gauge needle
  • 100% EtOH
  • 70% EtOH
  • DEPC water

RNA analysis

  • Quality control (RNA 6000 Nano Series II kit, Agilent Technologies, Santa Clara, CA, Cat#5067–1511)
  • DEPC water

Fluorescent labeling

  • cDNA labeling (CyScribe cDNA Post Labelling Kit, GE Healthcare, Little Chalfont, UK, Cat#RPN5660).
  • cDNA purification (CyScribe GFX Purification Kit, GE Healthcare Cat#27-9606-01).
  • λpolyA+ RNA-A (Takara Bio Inc., Otsu, Japan, Cat#TX802. prepared 1ng/μL)
  • 2.5 M NaOH (Prepared immediately before use, NaOH 100mg/1mL=2.5 M NaOH)
  • 0.1 M Sodium Bicarbonate (pH 9.0) : pH needs to be checked before use
  • 4 M Hydroxylamine Hydrochloride
  • chloroform / isoamyl alcohol (24:1)
  • 3 M sodium acetate, pH 5.2
  • 100% EtOH
  • 80% EtOH
  • 70% EtOH
  • Human or Mouse Cot-1 DNA (prepared 1 μg/μL): Human Cot-1 DNA (Invitrogen/Life Technologies, Carlsbad, CA, Cat#15279-011), Mouse Cot-1 DNA (Invitrogen/Life Technologies Cat#18440-016)
  • Yeast tRNA (Invitrogen/Life Technologies Cat#16401-011) prepared 1 μg/μL
  • Poly (dA) (GE Healthcare Cat#25-7836-02) prepared 1 μg/μL

DNA microarray

  • RIKEN Frontier Human 1K Glyco-gene Array ver.3
  • RIKEN Frontier Mouse 1K Glyco-gene Array ver.1

Hybridization

  • Takara Spaced Cover Glass L (Matsunami Glass Ind., Ltd., Osaka, Japan, Cat#TX703)
  • Pre-hybridization solution (6× SSC, 0.2% SDS, 5× Denhardt's solution, 2 mg/mL denatured salmon sperm DNA, filtrated with 0.22 μm membrane: These are 2 times concentrated than that of normally recommended.)
  • Hybridization solution (6× SSC, 0.2% SDS, 5× Denhardt's solution, 0.2 mg/mL denatured salmon sperm DNA, filtrated with 0.22 μm membrane: These are 2 times concentrated than that of normally recommended.)
  • 2× SSC-0.2% SDS (stay warmed at 65˚C, 55˚C)
  • 0.05× SSC
  • 0.2× SSC
  • 2× SSC
Instruments

In addition to normal molecular biology set up, following instruments are required to set up DNA microarray experiments:

  • Agilent 2100 bioanalyzer
  • Chip Priming Station (Agilent Technologies, Cat#5065-4401)
  • Vortexer model MS2-S8/MS2-S9 (IKA) with chip adaptor (Agilent Technologies, Cat#5​0​6​5​-​9​9​6​6)
  • Affymetrix 428 array scanner
  • Tupperware (airtight container, which can be used in the water)
  • Falcon1012
  • 50mL Falcon tube (put some Kimwipe paper in the bottom)
  • Water bath; 2 sets
  • Heat block
  • Centrifuge (at room temperature and 4˚C)
  • Glass slide washer baquette; 2 baskets and 5 trays
Methods
1.

PolyA plus RNA preparation

Due to its importance, we suggest to follow manufacture’s protocol as much as possible during mRNA preparation. Sample cells have to be fresh (discarded almost all supernatant) or/and immediately deep-freezed. We use mTRAP midi kit from Activemotif (USA), which directly enriches polyadenylated RNA with synthetic polyT probe. In some samples and tissue, preparation of total RNA prior to polyA enrichment is recommended. This could be due to the interference of polyA-T hybridization by the macromolecules present in cell lysates. Although high quality of mRNA preparation is very important, other system could offer equally effective preparation.

1) 

 Lyse cells with protease solution (1–3 mL) for 20–30 min at 45˚C.

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2) 

 Genomic DNA disruption with syringe.

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3) 

 Separate lysate into 1 mL in 1.5 mL tubes, and add 15 μL Poly T gripNA Probe to each tube.

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4) 

 After heating them at 75˚C, shake at room temperature

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5) 

 Add 60 μL Streptavidin Beads to each sample and stand for 45 min.

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6) 

 Centrifuge down the beads and wash them once.

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7) 

 DNase (RNase-free) treatment to degrade contaminating genomic DNA.

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8) 

 Wash beads twice.

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9) 

 Elute mRNA with DEPC-water twice and mix them.

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10) 

 Ethanol precipitation of mRNA.

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11) 

 Resuspend the pellet with 20 μL of DEPC-water.

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12) 

 Quantify RNA with A260 reading

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2.

Analysis of mRNA quality

Qualitative analysis of RNA is important thus should not be omitted. When polyA RNA is amplified, the same protocol of RNA analysis could be used. In amplified samples, peak of RNA will be shifted considerably toward smaller size. For comparison in DNA microarray experiments, use of similarly shifted amplified sample is recommended as the size of RNA may affect the labeling efficiency.

1) 

 Prepare 1 μL mRNA (concentration of 100~ ng/μL) preheated for 3 min at 75˚C.

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2) 

 Prepare a capillary electrophoresis cassette for RNA at Chip Priming Station.

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3) 

 Prepare the Gel-Dye mix and load to allocated well.

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4) 

 Load the marker (Agilent RNA 6000 Nano marker, Agilent Technologies) to all sample wells.

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5) 

 Load RNA samples to each lane and 1 μL RNA ladder (Agilent RNA 6000 Ladder, Agilent Technologies, Cat#5067-1529).

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6) 

 Vortex the chip.

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7) 

 Run on Bioanalyzer 2100 for 30–60 min.

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8) 

 Check for RNA degradation and ribosomal RNA (rRNA) contamination.

Contaminated rRNA should be deducted from RNA quantification data to adjust the amount of RNA used in labeling procedure.

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3.

Fluorescent cDNA labeling

It is advised to use polyA RNA free from rRNA. However, minor contamination of rRNA cannot be avoided in reality. Thus, adjusting polyA RNA by deducting rRNA is important. However, in general, we usually use polyA RNA samples less than 15% of rRNA contamination.

1) 

 Prepare RNA solution (1 μg ).

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2) 

 Add primer mix and heat at 70˚C.

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3) 

 Cool for 10 min at room temperature and add labeling mix.

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4) 

 Incubate for 45 min at 42˚C for reverse transcribe mRNA.

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5) 

 Purification of amino allyl-modified cDNA.

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6) 

 Coupling reaction for 90 min at room temperature in the dark for CyDye-labeling. While samples are coupled, pre-hybridization step of DNA microarray should be started.

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7) 

 Purification of CyDye-labeled cDNA.

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8) 

 Mix Cy3-labeled cDNA and Cy5-labeled cDNA (= 1 volume).

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9) 

 Add 1 volume of chloroform / isoamyl alcohol (24:1) and mix well.

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10) 

 Centrifuge and transfer upper layer to new tube.

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11) 

 Add 2.2 μL each of Cot-1, Yeast tRNA and Poly (dA) as competitors for hybridization noise.

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12) 

 Ethanol precipitation of labeled cDNA.

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13) 

 Check the color of labeled material in ethanol precipitate.

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14) 

 Resuspend labeled cDNA with hybridization solution.

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4.

DNA microarray

DNA microarray experiments require even hybridization regardless of the position of spot on glass slides. Thus, complete mixing of all reagents and tightly controlled temperature management is very important to avoid local concentration of reagents. Special care should be taken to achieve this.

1) 

 Pre-hybridize the microarray.

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2) 

 Place a Spaced-cover glass on DNA microarray, that is placed on top of Falcon1012 and load pre-hybridization solution to its interspace.

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3) 

 Put the top on Falcon1012, set them with wet Kimwipe paper in the Tupperware for 2 h~ at room temperature.

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4) 

 Slowly draw back the cover glass in the Glass slide washer tray filled with 2× SSC at room temperature.

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5) 

 Rinse DNA microarray in the tray filled with 0.2× SSC.

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6) 

 Put DNA microarray into the padded 50mL-Falcon tube.

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7) 

 Centrifuge the tube without cap at 600 rpm for 5 min at room temperature.

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8) 

 Heat labeled cDNA solution for 2 min at 95˚C and cool at room temperature in the dark.

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9) 

 Place a new Spaced-cover glass on the DNA microarray placed on Falcon1012 and load labeled cDNA solution to its interspace.

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10) 

 Put the top on Falcon1012, set them with wet Kimwipe paper in the Tupperware into plastic bag.

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11) 

 Sink them into the water bath for 16 h~ at 65˚C in the dark for hybridization. To keep them submerged in water, put three Glass slide washer trays filled with 2× SSC-0.2% SDS or water on them.

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12) 

 Slowly draw back the cover glass in the Glass slide washer tray filled with 2× SSC at room temperature.

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13) 

 Wash DNA microarray in the Glass slide washer tray filled with 2× SSC-0.2% SDS for 5 min at 55˚C twice.

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14) 

 Wash with 2× SSC-0.2% SDS for 5 min at 65˚C.

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15) 

 Rinse with 0.05× SSC at room temperautre.

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16) 

 Put DNA microarray into the padded 50mL-Falcon tube.

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17) 

 Centrifuge the tube without cap at 600 rpm for 5 min at room temperature.

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18) 

 Read fluorescence with microarray laser scanner.

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19) 

 Adjust fluorescence intensity between two channels while reading.

Comment 1
20) 

 Mark inappropriate spots to avoid noise in subsequent statistical analysis.

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Notes

Methods-1)

  • In the case of tissues, you may need to mince them in RNAlater (Applied Biosystems/Life Technologies Cat#AM7020) to avoid unwanted RNA degradation. Minced tissues are rinsed twice with RNase-free PBS and total RNA is isolated from them with your usual method. Thereafter, mRNA is isolated from total RNA with mTRAP total kit (Activemotif Cat#23012).
  • Yields may vary due to the type of sample, its developmental stage, growth conditions used etc. Yield of mRNA is 10–20 mg from 0.5–1.0 × 107 cells (animal tissue; 50–200 mg) in easier samples.
  • RNA sample can be amplified to obtain enough material suitable for labeling. Normal amplification experiments start with the use of 1 μg total RNA. Use of degraded sample for amplification should be avoided because amplification procedure shortens the RNA further.

Methods-2)

  • Good polyA RNA shows a smear signal which peaks between two rRNA bands. Thus relative mobility should be used for checking.

Methods-3)

  • Properly labeled sample has color incorporation in the precipitate. This could serve to judge the reaction. If no color is found in DNA precipitate, it does not worth to operate following DNA microarray hybridization.

Methods-4)

  • A good microarray result still has a false rate of 1% as marked inappropriate. Duplicated spots on a single glass slide would help to measure some expression of all the genes spotted.
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