DNA microarray
DNA microarray is used to monitor expression of large set of genes at the same time. In essence, DNA microarray is a platform carrying several tens of thousands of genes as DNA fragments on the certain location of a basement. Fluorescently labeled-cDNA synthesized from mRNA sample is hybridized to array to simultaneously determine the abundance of each gene. There are two types of DNA microarray, that differ by a type of DNA arrayed. One is PCR-amplified-cDNA type which tends to give stronger signal but have difficulty to prepare. The other is oligo-nucleotide type, whose hybridization signal can be weak but better coverage of genes could be easily achieved. Lately, oligo-nucleotide type DNA microarray is more popular between two due to the broader coverage in accordance with the progress in genome analysis. DNA microarray with cDNA can be useful to obtain transcriptome of a set of focused genes, such as glycan-related genes1) 2). Here we describe how we execute DNA microarray experiments using glycan-related gene microarray developed in RIKEN frontier research system 3). Since it is unlikely to read this protocol to actually perform microarray experiments elsewhere, we put more effort to describe information how readers could set up microarray experiments.
What can be achieved in DNA microarray experiments?
DNA microarray experiments normally detect differences in gene expression. Thus, it is important to judge if your gene of interest can be regulated by the transcriptional level. If gene product of interest is regulated by the post-translational modification such as phosphorylation, it is less likely that expression difference found from microarray experiments does not have much biological significance. Glycosyltransferase is a family of enzymes whose expression tends to determine the cellular enzymatic activity. Lectins tend to be regulated by the transcriptional level thus it can be effectively screened by DNA microarray experiments.
Quantitative vs. qualitative analysis
DNA microarray experiments use DNA hybridization technique to a vast number of spots on rather wider space, such as glass slides. Therefore, it is inevitable that experimental variations could be also detected in experiments. It is therefore important to validate the data in the context of fold-difference in relation with expression level. A 1.5-fold induction of a strongly expressed gene can be reliably measured by DNA microarray. However, detection of a 3-fold increase of a weakly expressed gene can be difficult. Weighing available information is essential for researchers to analyze high-volume data set obtained in “-omics” experiments.
MIAME
Although transcriptomic results obtained from DNA microarray data are informative, it is rather difficult to be utilized by other researchers when experimental set up is different between experiments. To achieve generalized set up within the community, Minimal Information About Microarray Experiment (MIAME) was created. Researchers should to follow MIAME recommendations when setting up DNA microarray experiments. Following these rules not only make DNA microarray result useful to others, it is required to deposit microarray data into database such as GEO. Since prior to submission the deposition of microarray data is required for publication in many scientific journals, it is essential to consult with MIAME before setting up microarray experiments.
Quality control of the experiments
One of the most important issues to obtain reliable signal in DNA microarray experiment is a quality of mRNA analyzed. Since it is vital, having high quality mRNA even exceeds the merit of duplicating the experiment. Therefore, it is strongly advised to quality check the mRNA sample prior to label for hybridization. Capillary electrophoresis devices could serve nicely to check the mRNA since low-quality mRNA tends to degrade in a variable degree and/or contains massive amount of ribosomal RNA contamination. Since RNA quantification involves A260 reading and ribosomal RNA exhibits the same ultraviolet absorption, deduction of ribosomal RNA from the calculation in labeling experiments is equally important to not having degraded RNA, the obvious problem frequently happens due to inappropriate handling of cells or tissues. Agilent 2100 bioanalyzer could be used for such purpose. We often see very different quality of mRNA even they are purchased from commercial vendors. Since quality of mRNA sample has a strong impact on the microarray experiments, it is also important to have similar quality of RNA used for biological replicate. Otherwise, replication of experiments simply confuses the interpretation of data.
Reverse transcription-mediated labeling of mRNA and hybridization
Since fluorescent dye-conjugated deoxynucleotide could cause skewed incorporation, it is advised to use a less-biased system. In this protocol, we use two-step protocol for labeling. In the first step, each mRNA is converted into cDNA with amino allyl-dUTP. In the second step, each amino allyl-modified cDNA is individually labeled with different CyDye NHS-esters by coupling reaction. Cy3 and Cy5 are common fluorescent dyes for this purpose. Labeled cDNA is mixed together in equal proportions and hybridized on the same DNA microarray for competitive hybridization. After washing, the DNA microarray is imaged using a scanner, and the extent of binding cDNA is detected as fluorescent intensity. The ratio of red and green fluorescence intensity at each spot indicates the relative quantitative difference of the correspondence mRNA between two samples.
Data processing
Normalization of the DNA microarray signal is essential to avoid experimental noise. Mass normalization is usually first choice because expression of “constitutively expressed genes-so-they-said” varies in many circumstances. To compare signal difference between fluorescence detector channels, researchers should be aware of the significance in the resulted difference. In general, difference in strong signal is more reliable than that of weak ones. 2D Scatter plot analysis will give some idea on whether or not the difference between two channels would significant. Nevertheless, data should be processed with appropriate statistical analysis to assess its significance.
Understanding the data
Microarray experiments could be very powerful when all the things are considered and properly planed; transcriptome of glycan-related genes are obtained in single experiments. Appropriate data-mining should be unexpectedly informative. Systems biologic/bioinformatics-oriented data processing is recommended to fully appreciate obtained results, rather than focusing on the particular set of genes of interest. For example, microarray data can be cross-compared with multiple samples to produce glycan-related gene expression profiles when a standard RNA was employed to compare. Such profiles were very useful to screen rate-limiting steps in the biosynthetic pathway of glycans when statistical correlation was examined 4) 5). |
Category | Glycosyltransferases & related proteins |
Protocol Name | cDNA microarray experiment: set up and execution |
Authors
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Takematsu, Hiromu
*
Laboratory of Biochemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University
Fujinawa, Reiko
-, RIKEN ASI
*To whom correspondence should be addressed.
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Reagents
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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
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RNA analysis
- Quality control (RNA 6000 Nano Series II kit, Agilent Technologies, Santa Clara, CA, Cat#5067–1511)
- DEPC water
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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
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DNA microarray
- RIKEN Frontier Human 1K Glyco-gene Array ver.3
- RIKEN Frontier Mouse 1K Glyco-gene Array ver.1
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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
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Instruments
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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#5065-9966)
- 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
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Methods |
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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.
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Lyse cells with protease solution (1–3 mL) for 20–30 min at 45˚C. |
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Genomic DNA disruption with syringe. |
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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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After heating them at 75˚C, shake at room temperature |
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Add 60 μL Streptavidin Beads to each sample and stand for 45 min. |
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Centrifuge down the beads and wash them once. |
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DNase (RNase-free) treatment to degrade contaminating genomic DNA. |
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Elute mRNA with DEPC-water twice and mix them. |
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Resuspend the pellet with 20 μL of DEPC-water. |
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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.
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Prepare 1 μL mRNA (concentration of 100~ ng/μL) preheated for 3 min at 75˚C. |
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Prepare a capillary electrophoresis cassette for RNA at Chip Priming Station. |
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Prepare the Gel-Dye mix and load to allocated well. |
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Load the marker (Agilent RNA 6000 Nano marker, Agilent Technologies) to all sample wells. |
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Load RNA samples to each lane and 1 μL RNA ladder (Agilent RNA 6000 Ladder, Agilent Technologies, Cat#5067-1529). |
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Run on Bioanalyzer 2100 for 30–60 min. |
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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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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.
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Add primer mix and heat at 70˚C. |
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Cool for 10 min at room temperature and add labeling mix. |
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Incubate for 45 min at 42˚C for reverse transcribe mRNA. |
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Purification of amino allyl-modified cDNA. |
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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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Purification of CyDye-labeled cDNA. |
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Mix Cy3-labeled cDNA and Cy5-labeled cDNA (= 1 volume). |
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Add 1 volume of chloroform / isoamyl alcohol (24:1) and mix well. |
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Centrifuge and transfer upper layer to new tube. |
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Add 2.2 μL each of Cot-1, Yeast tRNA and Poly (dA) as competitors for hybridization noise. |
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Ethanol precipitation of labeled cDNA. |
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Check the color of labeled material in ethanol precipitate. |
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Resuspend labeled cDNA with hybridization solution. |
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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.
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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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Put the top on Falcon1012, set them with wet Kimwipe paper in the Tupperware for 2 h~ at room temperature. |
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Slowly draw back the cover glass in the Glass slide washer tray filled with 2× SSC at room temperature. |
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Rinse DNA microarray in the tray filled with 0.2× SSC. |
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Put DNA microarray into the padded 50mL-Falcon tube. |
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Centrifuge the tube without cap at 600 rpm for 5 min at room temperature. |
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Heat labeled cDNA solution for 2 min at 95˚C and cool at room temperature in the dark. |
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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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Put the top on Falcon1012, set them with wet Kimwipe paper in the Tupperware into plastic bag. |
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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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Slowly draw back the cover glass in the Glass slide washer tray filled with 2× SSC at room temperature. |
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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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Wash with 2× SSC-0.2% SDS for 5 min at 65˚C. |
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Rinse with 0.05× SSC at room temperautre. |
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Put DNA microarray into the padded 50mL-Falcon tube. |
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Centrifuge the tube without cap at 600 rpm for 5 min at room temperature. |
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Read fluorescence with microarray laser scanner. |
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Adjust fluorescence intensity between two channels while reading. |
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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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Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-11-19 16:55:06 |
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Fujinawa, Reiko,
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cDNA microarray experiment: set up and execution.
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