Early gene expression microarrays consisted of arrayed spots each containing a cDNA corresponding to a single gene. For example, one of the first yeast gene expression microarrays consisted of about 6000 spots composed of cDNA corresponding to nearly every gene in the Saccharomyces cerevisiae genome (^{DeRisi et al. 1997}). cDNA microarrays have fallen into disuse because of the advent of oligonucleotide microarrays. Advances in oligonucleotide synthesis make it possible to generate gene expression microarrays composed of long oligonucleotides that have been designed to be complementary to a given gene. Oligonucleotides can be synthesized and sold in soluble form, which are then arrayed by a spotting robot, as with cDNA arrays. Alternatively, several commercial microarray suppliers use in situ oligonucleotide synthesis, in which oligonucleotides are printed directly onto the microarray substrate. This process allows for much more flexible generation of oligonucleotide microarrays and, thus, is used more often than soluble oligonucleotide arrays because a laboratory need not purchase an excess of oligonucleotides in order to run microarray experiments.

A typical mRNA expression experiment begins with the isolation of poly(A) RNA from two related populations of cells, such as yeast growing at 30C and yeast that have been shifted from 30C to 37C. Fluorophore-labeled cDNA is prepared from each mRNA population. Typically, the cyanine dyes used are Cy5 (pseudo-colored red in microarray images) and Cy3 (pseudo-colored green). The labeled cDNAs are mixed and hybridized to a microarray. After hybridization and washing, the microarray is scanned with lasers that excite the fluorophores, and the image is processed, generating a Cy5/Cy3 ratio for every spot on the array. The microarray data are normalized, and a list of the Cy5/Cy3 ratios per gene is generated, which can be analyzed for genome-wide changes in the transcriptome.

The microarray data are normalized so that the average log₂(Cy5/Cy3) is 0; this is a necessary step because the absolute intensity of either channel (Cy5 or Cy3) is subject to a wide range of modifying influences, such as input RNA level and labeling efficiency, among others. This normalization should always be kept in mind in interpreting microarray studies. For example, microarray results for a comparison between liver RNA and sperm RNA populations might indicate that a number of RNAs are relatively enriched in sperm, but the absolute numbers of those RNAs per nucleus are still probably lower in sperm than in liver given the disparity in RNA abundance in the two types of cells.

The experiment described above is capable of providing information about the relative abundances of every mRNA expressed in the two cell populations. What about absolute expression levels? These can be assayed in two ways. First, single-color experiments using the Affymetrix platform provide a reasonable measure of mRNA abundancethe intensity of a given spot is related to the absolute abundance of its complementary mRNA in the original labeled population. Second, for two-color experiments, a straightforward way to estimate transcript abundance is to compare labeled mRNA with labeled genomic DNA, which, in principle, should be present in a single copy per gene (unless the gene in question comes from a multicopy family). Thus, gDNA is labeled with Cy3, mRNA is labeled with Cy5, and Cy5/Cy3 ratios provide a relative measure of mRNA abundance. These ratios can be converted to absolute mRNA abundance if the absolute abundance of any RNA is already known. Normalization can be used to set the absolute level of these RNAs, and all remaining RNA abundances can be inferred from their Cy5/Cy3 ratios relative to the Cy5/Cy3 ratios for the known standards.

Comparative genomic hybridization (CGH) is used to detect changes in DNA copy number across a genome (^{Pollack et al. 1999}). In a CGH experiment, the two populations of genomic DNA are fluorescently labeled using Klenow DNA polymerase. Labeled DNA is compared using the same experimental design as for analysis of mRNA expression. Changes in copy number are reflected in the Cy5/Cy3 ratios. This method has been used most commonly in the study of tumors, where there is a well-documented role both for gene loss and for amplification in the process of oncogenesis. By analyzing changes in gene copy number, regional amplification or even whole chromosome copy-number changes have been characterized. Variants of this approach have also been used to characterize replication timing, as DNA isolated during S phase shows variations in copy number, based on whether or not the segment of genomic DNA has replicated.

Although changes in mRNA levels can be analyzed easily using microarrays constructed with one probe (spot) per gene, different methods are required to detect variations in transcript structure. These variations include changes in splicing patterns and in the start and stop sites for transcription. These and other changes in transcript structure can be detected and mapped using tiling microarrays. For example, transcriptional start sites can be mapped to 1020-nucleotide resolution by a variant of the absolute abundance hybridization method described above. Briefly, mRNA is labeled with Cy5 and hybridized against Cy3-labeled genomic DNA. In principle, an expressed RNA should appear as a long square wave of high Cy5/Cy3 ratios: Cy5/Cy3 ratios should be low in spots corresponding to genomic sequences that flank those complementary to the RNA. High Cy5/Cy3 ratios should start at spots containing sequences corresponding to the 5 end of the RNA and should continue to the end of the RNA (Fig. 2). Indeed, this technique has been used to determine transcriptional start sites to 20-nucleotide resolution in yeast (^{Yuan et al. 2005}) and to detect and map novel transcripts in human cells (^{Shoemaker et al. 2001}).

Variations in splicing patterns can also be detected and mapped using microarrays. With tiling arrays, expressed exons should appear as peaks of Cy5/Cy3. Another way to determine exon inclusion is to use an exon microarray, in which each spot on the array corresponds to a single exon. When probing an exon microarray, for a given tissue all of a gene's expressed exons will show high Cy5/Cy3 (^{Shoemaker et al. 2001}).

Tiling microarrays have also been used to discover noncoding RNAs and novel genes. The reason for this is obviousa classical, expression microarray only covers what is known, whereas a tiling microarray can be designed to contain an entire genome. For example, tiling microarrays of chromosomes 21 and 22 revealed the nearly pervasive low-level transcription of the human genome (^{Kapranov et al. 2002}).

RNA molecules associated with specific RNA-binding proteins have been identified using immunoprecipitation assays to isolate a protein of interest. The associated RNA molecules (or regions of RNA) are then compared with nonspecific RNAs that have been isolated in control immunoprecipitations lacking antibody (^{Hieronymus and Silver 2003}; ^{Gerber et al. 2004}).

The functional properties of RNAs (e.g., translation) can be assayed by microarray using more sophisticated fractionation techniques. In an early study, polysomal RNAs were analyzed by microarray techniques (^{Arava et al. 2003}). Subcellular localization of RNAs can also be studied when appropriate purification techniques exist; for example, isolation of dendrites from neurons has been used to identify the relevant RNA populations (^{Eberwine et al. 2002}).

At present, one of the most popular applications of tiling microarrays is known as chromatin immunoprecipitation on chip (or ChIP-on-chip; see Chapter 20). By this method, the location of a specific protein, such as a transcription factor, is assayed through the use of chromatin immunoprecipitation. Briefly, proteins are covalently cross-linked to the genome using formaldehyde, and chromatin is sheared by sonication to small (500 bp) fragments. Using an antibody specific to a given protein (with either an antibody to an epitope tag, a protein-specific antibody, or even an antibody against a specific modification state), DNA associated with the protein in question is isolated by immunoprecipitation. After washing, the cross-links are dismantled and genomic DNA is isolated. The DNA is typically amplified, and the amplified material is labeled and hybridized competitively against labeled amplification reactions from no-antibody controls or from pre-IP material. Analysis of ChIP-on-chip microarray results provides a snapshot of a transcription factor's location on the genome and has proven to be a tremendously powerful method for studying transcriptional regulation by transcription factors and chromatin (^{Ren et al. 2000}; ^{Iyer et al. 2001}).

Tiling microarrays can also be hybridized with genomic DNA that has been treated diagnostically with a nuclease before labeling the DNA. This method takes advantage of the fact that chromatin packaging of the genome dramatically affects nuclease accessibility. Two nucleases have been used in genome-wide studies. First, DNase Ihypersensitive sites have long been known to occur at genomic regulatory elements such as promoters, enhancers, and insulators. Here, chromatin is lightly digested with DNase I, sites of cleavage are isolated, and DNA surrounding cleavage sites is interrogated by tiling microarray analysis (^{Sabo et al. 2006}). Second, micrococcal nuclease (MNase) is a nonprocessive nuclease with preference for the linker DNA between nucleosomes. Analysis of the DNA protected from MNase cleavage including comparison to whole genomic DNA has proven to be an invaluable tool in genome-wide nucleosome mapping studies (^{Yuan et al. 2005}).

Although exon microarrays (see above) can monitor expression of each exon represented on the array, the structure of the transcript cannot be discerned. Specifically, exon connectivity is not detected using exon microarrays. For example, expression of exons 1, 2, 3, and 5 could indicate a single species containing all four exons, or two or more distinct species (1-2-5 and 1-3-5, etc.). To determine connectivity of exons requires the use of splicing microarrays, which are comprised of oligonucleotides complementary to specific splice junctions.

The sequence specificity of DNA hybridization has also been leveraged for genome analyses such as resequencing and polymorphism detection. In these applications, each genomic location is represented on the microarray by several oligonucleotides that differ by a single nucleotide. In other words, if the genomic region of interest has the sequence AATGCCA, oligonucleotides containing AATTCCA, AATCCCA, and AATACCA will also be printed. Hybridization of genomic DNA to these oligonucleotides can be used to determine mutations or sequence polymorphisms at the site interrogated by the set of oligonucleotides.

One way to use microarrays for resequencing is the so-called sequence capture protocol (^{Hodges et al. 2007}). A microarray is printed with oligonucleotides corresponding to genomic regions of interest. DNA or RNA is hybridized to the microarray, and all complementary sequences are retained on the microarray through the washing protocols. After retention of desired genomic regions via hybridization, this hybridized material is eluted and sequenced using high-depth sequencing methods (Chapter 11).

A great variety of microarrays are commercially available, and for most researchers, an off-the-shelf product will suffice. If not, customized microarrays can be ordered from several manufacturers or designed (and printed) in-house. The appropriate design of a microarray will depend on the application for which it is to be used. There are, however, some basic design principles that are common to most applications. We consider two types of basic microarray: the gene expression microarray and the tiling microarray. Specialized formats, such as splicing arrays and resequencing arrays, have been designed for different organisms, and we point interested readers to the relevant literature (^{Hacia 1999}; ^{Mockler et al. 2005}; ^{Blencowe 2006}; ^{Hughes et al. 2006}; ^{Calarco et al. 2007}; ^{Cowell and Hawthorn 2007}; ^{Gresham et al. 2008}).

Once oligonucleotides have been designed, microarrays can be printed commercially. Alternatively, oligonucleotides can be synthesized commercially or by an in-house core facility, and then printed in-house using a spotting robot (see Protocol 1).

Most of the sample preparation procedures for microarrays follow standard protocols, many of which can be found elsewhere in this manual. For example, comparative genomic hybridization (CGH) analyses use genomic DNA isolated from samples of interest, as described in Chapter 1. Gene expression or splicing studies use either total RNA or mRNA, purified as described in Chapter 6. Protein localization analysis starts with material isolated via chromatin immunoprecipitation (ChIP), as described in Chapter 20. Typically, however, the intended assays for many of these protocols are based on blotting techniques or quantitative PCR readouts, which often require only nanograms of material. Microarray labeling, on the other hand, typically requires several micrograms of nucleic acid; therefore, an amplification step is necessary before labeling.

Labeling nucleic acids for use in microarrays is similar for most microarray platforms, except for Affymetrix microarrays. For most platforms (homemade or commercial), fluorescent molecules are attached to DNA by the Klenow fragment of DNA polymerase I. For labeling RNA, reverse transcriptase is used to prepare labeled cDNA. Labeling methods can include a fluorescent dNTP in the labeling reaction (Protocols 5 and 7). Because this approach is rapid but expensive, a cheaper but lengthier alternative first incorporates aminoallyl nucleotides into the nucleic acid molecules and then couples the aminoallyl group to the fluorophore (Protocols 6 and 8). In all of the labeling protocols, the source nucleic acids can be either unamplified or amplified before labeling.

Methods for hybridization of labeled probe materials to a microarray differ significantly when using home-printed microarrays versus commercial microarrays. For home-printed microarrays, slides must first be blocked because polylysines remaining on the slide will cause significant background binding to labeled material unless they are neutralized by reaction with succinic anhydride. Protocols 9 and 10 provide methods, respectively, for blocking and hybridizing to homemade arrays. When using commercial microarrays, hybridization protocols are typically provided. Following hybridization, microarrays are scanned in a bench-top scanner. The data are collected, formatted, and stored digitally for subsequent analysis.

The tools used for analysis of microarray data will depend on the experimental question being asked: Localization studies require a different set of tools than do gene expression studies. Some of the available resources are described in Chapter 8. Here, we outline some of the basic steps in data analysis.

The first step in microarray data analysis is to remove bad data (i.e., data from spots that were flagged because they were obscured by fluorescent precipitate, etc.), and to normalize the remaining data. Working with a .gpr file, which is the standard output of GenePix software, we typically eliminate all flagged probes and then work exclusively with the Log₂ ratio data. More advanced users may consider using specific features, such as Foreground and Background intensities.

Most two-color microarray studies are normalized to an average Log₂ ratio value of 0. The assumption implicit in this normalization is that there was no overall change in whatever is being measured. Thus, it is important to remember when looking at such normalized data that relative values are being measured, not absolute values. Practically, normalization can be performed by averaging the unflagged log ratio value, then subtracting this value from every one of the entries in the column. This can be done by hand in spreadsheet programs or using common commands in languages such as MATLAB, Perl, or R.

Once normalization is completed, the list can be sorted by log ratio value to identify genes that are dramatically up-regulated or down-regulated (if gene expression) or identify loci with high levels of enrichment of the protein in question. At this point, data analysis paths will diverge depending on the questions being asked. However, because many microarray studies involve multiple microarrays, it is often useful to cluster data to identify genes or loci that share similar behavior.

For clustering and visualization, numerous programs are available online. We use the classic Cluster and TreeView programs (^{Eisen et al. 1998}), available online at SourceForge (http://sourceforge.net/) or via the Eisen laboratory website (http://www.eisenlab.org/). Because sample files that guide the formatting of files for clustering are also available, formatting will not be described here. Briefly, however, data will be loaded into Cluster, various thresholds will be set (fraction of data missing for a given gene, number of genes changing over some threshold, etc.), and one of several clustering algorithms will be used. The output of the clustering can be visualized with TreeView, allowing users to generate the classic heatmap view of their microarray data.

• Oligonucleotides of desired sequences
  • Oligonucleotides are typically prepared at 2040 m in aqueous 3 SSC.
• Salmon sperm DNA <A>, sheared (150 ng/L resuspended in 3 SSC)
• SSC (3) <A>

• Desiccator
• Nitrogen gas, compressed
• Plates (384 wells)
• Polylysine-coated slides
  • Although polylysine coating can be done in-house, in our experience, the failure rate is significant and purchasing commercial polylysine-coated slides (e.g., Erie Scientific) has proven to be the most cost-effective solution.
• Slide box, plastic
• Spotting robot

• 10. Start the print run. Keep careful notes about plate order and orientation. Knowing the plate order is necessary for creating the .gal file that maps spot position to gene name when microarray data are analyzed. Any mistakes during printing such as printing plates out of order (e.g., plate 3 before plate 2) can be corrected later on as long as they are noted.
• 11. When a plate is finished printing and the print head has come to a complete stop, either let the plate evaporate in a hood if the plates are stored dry, or cover the plate with foil and its lid and store the plate at 80C.
• 12. Insert the next print plate into the plate holder.
• 13. When the print run is done, let the slides dry overnight, unless you are performing back-to-back print runs.

• aa-dNTP/Cy-dNTP mixture (100) <R>
• BSA (500 g/mL)
• DNA, isolated from the sample under study (10100 ng)
  • For example, for CGH analysis, genomic DNA is isolated as described in Chapter 1; for protein localization studies, DNA is isolated by chromatin immunoprecipitation (ChIP) as in Chapter 20.
• dNTP mixture (3 mM)
• dNTPs (100; 20 mM each nucleotide)
• DTT (0.1 M)
• MgCl₂ (25 mM)
• PCR buffer (10) (500 mM KCl, 100 mM Tris at pH 8.3)
• Primer A: GTTTCCCAGTCACGATCNNNNNNNNN (40 pmol/L)
• Primer B: GTTTCCCAGTCACGATC (100 pmol/L)
• Sequenase (13 units/L) (US Biochemical, catalog no. 70775)
• Sequenase buffer (5)
• Sequenase dilution buffer
• Taq polymerase (5 units/L)

• Agarose gel (1)
• Microcon 30 spin column (Millipore)
• Thermal cycler

• 1. Prepare Round A reactions as follows:
  • As little as 10 ng of DNA can be effectively amplified by this protocol. As a control, set up a reaction in which the DNA is replaced with H₂O.
• 2. Denature the template DNA and anneal the primer by heating for 2 min at 94C and then rapidly cooling to 10C. Keep the reaction for 5 min at 10C.
• 3. Assemble the reaction mixture:
• 4. Combine the reaction mixture and the templateprimer mix. Place the tube(s) into a thermal cycler, and extend the primers as follows.
  • i. Ramp from 10C to 37C over 8 min.
  • ii. Hold at 37C for 8 min.
  • iii. Rapidly ramp to 94C and hold for 2 min.
  • iv. Rapidly ramp to 10C, add 1.2 L of diluted Sequenase (1:4 dilution), and hold for 5 min at 10C.
  • v. Ramp from 10C to 37C over 8 min.
  • vi. Hold at 37C for 8 min.
• 5. Dilute samples with water to a final volume of 60 L.

• 6. Prepare Round B reactions as follows:
• 7. Place the tube(s) into a thermal cycler, and amplify the templates as follows.
  Run 1535 cycles, depending on the amount of starting material.
  • To optimize the number of cycles, it may be necessary to remove an aliquot every two cycles to monitor the progress of the amplification. It is best to use the minimal number of cycles that generates a visible smear (see Step 8).
    Make sure that there is no DNA in the negative control lane!
• 8. Run 5 L of the reaction on a 1 agarose gel. A smear of DNA should be present between 500 and 1000 bp.
• 9. Label the DNA using the Round C procedure, Protocol 7, or Protocol 8.

• 10. Prepare the Round C reaction:
• 11. Place the tube(s) into a thermal cycler, and label the templates as follows:
  Run 1025 cycles.
  • The number of cycles should be determined empirically. The objective is to minimize the number of cycles required to yield 23 g of material for hybridization.
• 12. If aa-dNTPs were used in Round C, desalt the sample to remove Tris buffer, which interferes with the dye coupling. Add 400 L of water to the sample in a Microcon 30, and centrifuge for 8 min at 12,000 rpm. Repeat again with 500 L of water.
• 13. Proceed to Cy-dye coupling as described in Protocol 8, Step 11.

• -Mercaptoethanol
• Calf intestinal phosphatase (CIP) (New England Biolabs, catalog nos. M0290S or M0290L)
• CoCl₂ (5 mM)
• Dideoxynucleotide tailing solution (8) (92 mM dTTP, 8 mM ddCTP) (Life Technologies)
• dNTP mixture (5 mM) <A>
• EDTA (0.5 M, pH 8.0) <A>
• Ethanol (95100)
• Klenow fragment of DNA polymerase I (New England Biolabs)
• Mineral oil
• NEB buffer 2 (10) (New England Biolabs)
• NEB buffer 3 (10) (New England Biolabs)
• NTP mixture (75 mM)
• Reaction buffer, provided with kit
• RNase-free H₂O
• RNase inhibitor
• T7-A₁₈B primer
  • The primer sequence is 5-GCATTAGCGGCCGCGAAATTAATACGACTCACTATAGGGAG(A)₁₈[B], where B refers to C, G, or T. The primer should be HPLC, PAGE, or equivalent purification grade.
• T7 RNA polymerase
• Template DNA (maximum 500 ng per 10 L)
• Terminal transferase (TdT) (New England Biolabs, catalog nos. M0315S or M0315L)
• Terminal transferase buffer (5) (Roche, catalog no. 11243276103)

• MinElute kit (QIAGEN, catalog no. 28204)
• RNase/DNase-free tubes (1.5 mL)
• RNase-free PCR tubes (0.2 mL)
• RNeasy Mini Kit (QIAGEN)
• Rotary evaporator (e.g., SpeedVac)
• Thermal cycler
• Vacuum manifold (optional; see Step 17)
• Water bath or heat block set to 37C

Treatment of DNA with calf intestinal phosphatase (CIP) is only necessary if the source DNA has been sheared or treated with MNase. Treatment of the DNA with CIP removes 3-phosphate groups, leaving free hydroxyl groups on the 3 ends, which are necessary for efficient tailing by TdT. Failure to perform this step will likely reduce the yield of amplification products by 50.

• 1. Set up the following reaction:
  Incubate the reaction for 1 h at 37C.
  • Each reaction can be scaled up to 100 L per tube.
• 2. Clean up the DNA with a MinElute column. Follow the protocol supplied by the manufacturer. Elute the DNA in 20 L.
  • When working with <100 ng of DNA, the 10-L elution volume in the manufacturer's protocol may yield less than the 80 claimed by QIAGEN. Increase the elution volume to 1520 L, and reduce the volume of the DNA by drying, if necessary.

• 3. Set up the tailing reaction:
  • Do not use the NEB buffer 4 supplied with the NEB terminal transferase because DTT in the buffer will precipitate the CoCl₂ and inhibit the reaction. Use the cacodylate buffer (1 M potassium cacodylate, 125 mM Tris-HCl, and 1.25 mg/mL BSA at pH 6.6), either supplied with the Roche enzyme or purchased separately. Take precautions in handling this arsenic-containing buffer and use waste-disposal practices appropriate for your institution.
  • Do not freeze and thaw the dNTP mixes more than three times. Additional freezethaw cycles will degrade the dNTPs and will reduce the efficiency of the reaction.
  • Aim for 1 pmol of template molecules. The tested range is 2.575 ng of DNA per 10 L of reaction volume. Scale up the reaction volume accordingly for greater starting amounts. For ChIP samples, use a sensitive UV-Vis spectrophotometer or a fluorometer to quantify the amount of sample precisely. If the amount of DNA is unknown, scale up to a 20-L volume to ensure that there is enough TdT enzyme present for an efficient tailing reaction. Note that if insufficient enzyme is used, the efficiency of subsequent steps in the protocol will be significantly affected and result in significantly reduced yields (as little as 510 of normal expected yields).
  • We strongly suggest that the NEB terminal transferase be used for this protocol; TdT enzyme from other sources may not perform optimally. If using the Roche recombinant TdT, double the volume of enzyme.
• 4. Add 12 drops of mineral oil to the top of the mixture to prevent evaporation of reactants during incubation. Incubate the reaction for 20 min at 37C.
• 5. Stop the reaction by adding 2 L (per 10 L of reaction volume) of EDTA (0.5 M, pH 8.0).
• 6. Clean up the DNA with a MinElute column. Follow the protocol supplied by the manufacturer. Elute the DNA in 20 L.
  • If the starting volume is 10 L, then add 10 L of water to bring the volume to 20 L before adding the reaction to the spin column. When working with <100 ng of DNA, the 10-L elution volume in the manufacturer's protocol may yield less than the 80 claimed by QIAGEN. Increase the elution volume to 1520 L, and dry the volume down if necessary.

• 7. Set up the second-strand synthesis reaction:
  • If production of template-independent product is a significant problem, scale down the reaction volume while keeping the reagent concentrations (except for the T-tailed DNA) constant. See the end of the protocol for an example.
  • Do not freeze and thaw the dNTP mixes more than three times. Additional freezethaw cycles will degrade the dNTPs and will reduce the efficiency of the reaction.
  • NEB (early 2004) switched the supplied buffer for Klenow enzyme from EcoPol buffer to NEB buffer 2. This buffer should provide at least comparable yields to the old buffer and may actually increase yields up to 14.
  • Do not use mineral oil. Trace amounts of mineral oil appear to interfere with cleanup and in vitro transcription.
• 8. Use the following program in a thermal cycler:
  • i. 2 min at 94C.
  • ii. Ramp from 94C to 35C at 1C/sec, then hold for 2 min to anneal.
  • iii. Ramp from 35C to 25C at 0.5C/sec.
  • iv. Hold for 45 sec at 25C (or up to 6 min).
    • During this time, add 1 L (5 U) of Klenow DNA polymerase. If necessary, centrifuge the tube to remove condensation from the top and sides of the tube.
  • v. 37C, 90 min.
  • vi. (Optional) 4C to temporarily halt enzyme activity until user returns to take reaction tubes out of cycler.
• 9. Stop the reaction by adding 2.5 L of EDTA (0.5 M, pH 8.0) (the final concentration will be 45 mM).
• 10. Clean up the DNA with a MinElute column. Follow the protocol supplied by the manufacturer. Elute the DNA in 20 L.
  • When working with <100 ng of DNA, the 10-L elution volume in the manufacturer's protocol may yield less than the 80 claimed by QIAGEN. Increase the elution volume to 1520 L, and dry the volume down if necessary. An elution volume of 20 L at this step increased yields by 3040 for a 50-ng sample.

• 11. In vitro transcription (IVT) requires that the double-stranded DNA (dsDNA) be in an 8-L volume. Dry down the eluate from 20 to 8 L in a rotary evaporator at medium heat for 1012 min (the drying rate is 1 L/min).
• 12. Set up the in vitro transcription reaction in 0.2-mL RNase-free PCR tubes as follows:
  • If the IVT kit is new, combine the NTPs in one tube, then realiquot into four tubes. In the first three freezethaw cycles, yields drop 1015 after each cycle. If the NTPs go through more than three freezethaw cycles, each subsequent freezethaw cycle may drop the yield by as much as 50.
  • The buffer should be at room temperature. Adding cold buffer and dsDNA may cause the DNA to precipitate. If there is a precipitate, warm the buffer to 37C until the precipitate dissolves.
• 13. Incubate the reaction overnight at 37C in a thermal cycler with a heated lid or in an air incubator.
  • The incubation can range from 5 to 20 h; typical is overnight, roughly 16 h.

• 14. Prepare the buffer (433.5 L per IVT reaction):
• 15. Aliquot the mix into 1.5-mL RNase/DNase-free tubes.
• 16. Transfer the contents of the IVT mix (from Step 13) to the RNase/DNase-free tube, and vortex gently and briefly.
• 17. Add 250 L of 95100 ethanol, and mix well by pipetting. (Do not centrifuge!) Purify the aRNA through an RNeasy column by either the centrifuge method or with a vacuum manifold. aRNA Purification Using a Centrifuge
  • i. Apply the entire sample to an RNeasy Mini spin column mounted on a collection tube. Centrifuge the column for 15 sec at 8000g. Discard the flowthrough.
  • ii. Transfer the RNeasy column to a new 2-mL collection tube. Add 500 L of Buffer RPE (which must have ethanol added before use) and centrifuge for 15 sec at 8000g. Discard the flowthrough, but reuse the collection tube.
  • iii. Add 500 L of Buffer RPE onto the RNeasy column and centrifuge for 2 min at maximum speed.
  • iv. Remove the flowthrough, and pipette another 500 L of Buffer RPE onto the column. Centrifuge for 2 min at maximum speed.
    • This additional wash, which is not in the QIAGEN protocol, is necessary because of guanidinium isothyocyanate contamination in the eluted RNA.
  aRNA Purification Using a Vacuum Manifold
  • i. Apply the sample (700 L) to an RNeasy Mini spin column attached to a vacuum manifold. Apply vacuum.
  • ii. Shut off the vacuum, and pipette 500 L of Buffer RPE onto the RNeasy column. Apply vacuum.
  • iii. Repeat Step ii. Transfer the columns to 2-mL collection tubes. Centrifuge for 1 min at full speed.
  • iv. Return the column to the vacuum manifold, and add 500 L of Buffer RPE. Apply vacuum.
  • v. Transfer the column back to a 2-mL tube. Centrifuge for 1 min at full speed to completely dry the column.
• 18. Transfer the RNeasy column into a new 1.5-mL collection tube, and add 30 L of RNase-free water directly onto the membrane. Centrifuge for 1 min at 8000g to elute the RNA. Repeat if expected; the yield is >30 g.
• 19. Check the RNA concentration and purity by measuring the A₂₆₀ and A₂₆₀/A₂₈₀.
  • See Troubleshooting.
• 20. Proceed to Protocol 5 or 6 to add fluorescent label to the RNA.

Occasionally a low-molecular-weight band may also appear near the bottom of the gel, at 100 bp. It has been observed under certain amplification conditions, usually when the concentration of starting material is significantly less than that of the primer during second-strand synthesis. In these cases, a substantial amount of small-molecular-weight material may be generated, which likely represents amplification product produced from IVT-valid template synthesized through the formation of primer dimers during second-strand synthesis. These products represent nonproductive material for downstream analysis, and if substantial amounts of this material are observed, then it is necessary to limit the amount of primer.

Limiting the amount of primer is important when amplifying from very small amounts of starting material. Not only will it decrease the amount of primer-dimer product, it can also increase the yield of the desired amplification product. Table 2 describes the single reaction volumes to use for a suggested mass range of starting material.

• Carrier
  • Add either 5 g of linear polyacrylamide (LPA) or 20 g of glycogen. If a second round of amplification is to be used (or other downstream reverse transcription reactions), then LPA is recommended over glycogen. LPA will slow the Microcon washes (from 1214 min to 2832 min for Microcon 100s at 500g and room temperature), but it does not inhibit reverse transcriptase.
• DEPC ddH₂O
• DEPC-treated TE (pH 8.0)
• DNA ligase, from Escherichia coli
• DNA polymerase I
• dNTP (10 mM)
• DTT (100 mM)
• (dT)-T7 primer
  • The primer sequence is 5-GCATTAGCGGCCGCGAAATTAATACGACTCACTATAGGGAGA(T)21V-3 (where V stands for A, C, or G).
• Ethanol (70 and 95)
• First-strand buffer (5), provided with kit
• High-yield T7 in vitro transcription kit (e.g., AmpliScribe from Epicentre; similar kits from Ambion, Promega, and others are available)
• IVT buffer (10), provided with IVT kit
• NaCl (5 M)
• NTP mixture (100 mM)
  • The NTP mixture contains 100 mM each of ATP, CTP, GTP, and UTP.
• Phenol:chloroform
• Random primers
• Reverse transcriptase (SuperScript II; Life Technologies)
• RNase H, from E. coli
• RNase inhibitor
• Second-strand synthesis buffer (5) <R> (Lifetech or homemade)
• T4gp32 (single-strand binding protein; 8 mg/mL)
• T4 DNA polymerase
• T7 RNA polymerase, high concentration (80 U/L)
• Total RNA (100 ng dissolved in water or TE; the concentration does not matter as the RNA will be dried down in Step 1)

• Bio-Gel P-6 Micro-Spin Column (Bio-Rad)
• Heat block set at 65C and 70C
• Incubator set at 14C16C
• Microcon 100 spin column (Millipore)
• Phase Lock Gel Heavy tubes (0.5 mL) (Eppendorf)
• Thermal cycler (with a heated lid) or air incubator set at 42C and 70C
• Tubes (0.6 mL)
• Rotary evaporator (e.g., SpeedVac)

• 1. Combine 100 ng of the (dT)-T7 primer with 100 ng of total RNA in a 0.6-mL tube. Reduce the volume under vacuum to 5.0 L.
  • Do not allow the RNA to dry out completely.
• 2. Prepare the RT premix and place it on ice:
• 3. Denature the RNA/primer mix for 4 min at 70C in a thermal cycler with a heated lid.
• 4. Snap-cool the mix on ice and keep it on ice.
  • The volume may drop following denaturation.
• 5. Add 5.0 L of ice-cold RT premix to the RNA/primer tube, and mix by pipetting. The final volume should be 10 L.
  • If there was evaporative loss during denaturation (Step 3), then add dH₂O to adjust the volume to 10.0 L. Before committing your RNA, run these initial steps using a control nucleic acid to determine the volume loss.
• 6. Incubate the RT reaction for 1 h at 42C in either a thermal cycler with a heated lid or an air incubator, but not in a water bath to reduce the chance of contamination.
• 7. Heat-inactivate the reaction for 15 min at 65C.
• 8. Chill the tube on ice.

• 9. Prepare second-strand synthesis (SSS) premix:
  Chill on ice.
• 10. Add 65 L of ice-cold SSS premix to the RT reaction tube, and mix by pipetting. Incubate for 2 h at 14C16C.
• 11. Add 2 U (10 U) of T4 DNA polymerase, and mix by flicking and gentle vortexing. Incubate for an additional 15 min at 14C16C.
• 12. Heat-inactivate the reaction for 10 min at 70C. Go immediately from 15C to 70C without letting the tube sit at room temperature to avoid undesirable enzyme activities.
• 13. Add 75 L of phenol:chloroform (1:1), and mix by pipetting vigorously. Transfer the mixture to prespun Phase Lock Gel Heavy 0.5-mL tubes, and centrifuge for 5 min at 13,000 rpm.
• 14. Prepare a Bio-Gel P-6 Micro-Spin Column per the manufacturer's instructions.
• 15. Transfer the aqueous phase from Step 13 to the prepared P-6 column and centrifuge at 1000g for 4 min, recovering the flowthrough (80 L) in a clean 1.5-mL tube.
  • The flowthrough can be kept in the 1.5-mL tube or transferred to a tube of a different size, depending on the details of the IVT reaction steps. For example, it may be desirable to run the reaction in a thermal cycler; thus, the products can be held at 4C rather than overincubate them.
• 16. Add the appropriate carrier and 3.5 L of 5 M NaCl for precipitating the DNA, and mix by vortexing. Add 2.5 volumes of 95 ethanol (220 L) and mix well. Precipitate for at least 2 h at 20C.
• 17. Centrifuge to pellet the DNA at 13,000 rpm for 20 min.
• 18. Carefully remove the supernatant. Wash the pellet with 500 L of 70 ethanol, and centrifuge for 5 min at 13,000 rpm.
• 19. Carefully remove the supernatant. Pulse-centrifuge (up to full speed) the tube to collect all residual ethanol at the bottom.
• 20. Remove the remaining supernatant with a pipette. Allow the pellet to air-dry for 23 min.

• 21. Prepare the IVT premix at room temperature to avoid forming a precipitate:
• 22. Add 40 L of IVT premix to the DNA pellet (from Step 20). Resuspend the pellet in the premix by gently flicking and vortexing the tube. Incubate the reaction for 9 h at 42C.
  • If the starting amount of total DNA was in the microgram range, then IVT yields might improve by using a 60- or 80-L reaction volume.
• 23. Proceed with the cleanup and additional amplification, or freeze the IVT reaction at 80C.

• 24. Add 480 L of DEPC-treated TE to the IVT reaction tube.
• 25. Transfer the 500 L to a Microcon 100, and centrifuge at 500g until the volume is <20 L (1115 min at room temperature without LPA, 2832 min with LPA).
  • Processing a sample through a Microcon spin column is slower with LPA in the sample, although the results are fine. Alternatively, RNeasy columns can be used for cleanup.
• 26. Add another 500 L of DEPC-treated TE, and centrifuge as before. Repeat this step one more time (three washes total).
  • If you intend to proceed with a second amplification, the final wash should be with dH₂O, and with the filtrate should be reduced to a small volume.
• 27. Measure and, if necessary, adjust the volume for downstream applications.
  • It may be comforting or worthwhile to quantify the yield and analyze the products by electrophoresis. Alternatively, proceed to the second round of amplification.

• 28. Add 0.5 g of random primers to the aRNA (from Step 26). Reduce the volume under vacuum to 5.0 L.
• 29. Denature the RNA for 5 min at 70C in a thermal cycler with a heated lid.
• 30. Snap-cool the mix on ice. Let the tube rest for 5 min at room temperature.
• 31. Prepare the RT premix and place it on ice:
• 32. Add 5 L of room-temperature RT premix to the RNA, and mix by pipetting.
  • The final volume should be 10.0 L. If there was evaporative loss during denaturation (Step 28), then add dH₂O to adjust the volume to 10.0 L.
• 33. Incubate the reaction tube in a thermal cycler with a heated lid, programmed as follows:
  • i. 20 min at 37C.
  • ii. 20 min at 42C.
  • iii. 10 min at 50C.
  • iv. 10 min at 55C.
  • v. 15 min at 65C.
  • vi. Hold at 37C.
• 34. Add 1 U of RNase H, and mix by vortexing gently. Incubate for 30 min at 37C and then heat for 2 min at 95C.
• 35. Chill the tube on ice, and then centrifuge it briefly to collect the condensation. Place the tube on ice.
• 36. Add 1 L of 100 ng/L (dT)-T7 primer while the tube is on ice. Incubate the tube for 10 min at 42C to anneal the primer.
• 37. Prepare the SSS premix (minus ligase):
• 38. Snap-cool the sample (from Step 36) on ice.
• 39. Add 65 L of ice-cold SSS premix to the chilled reaction tube. Incubate for 2 h at 14C16C.
• 40. Add 10 U of T4 DNA polymerase, and mix by gentle flicking and vortexing. Incubate for an additional 15 min at 14C16C.
• 41. Heat-inactivate the reaction for 10 min at 70C.
• 42. Perform phenol:chloroform extraction, Bio-Gel P-6 chromatography, and nucleic acid precipitation as per Steps 1320.
• 43. Prepare the IVT premix as in Step 21.
• 44. Add 40 L of IVT premix to the DNA pellet (from Step 42). Resuspend the pellet in the premix by gently flicking and vortexing the tube. Incubate the reaction for 9 h at 42C.
• 45. Proceed with the cleanup as in Steps 2427, or freeze the IVT reaction at 80C.

• Cy3-dUTP or Cy5-dUTP (GE Healthcare Life Sciences)
• DEPC-treated H₂O
• HCl (0.1 N)
• NaOH (0.1 N)
• Oligo(dT) (2 g/L)
• Random hexamers (4 g/L)
  • N6 random hexamer can be ordered from any oligonucleotide company and made up at 5 mg/mL in RNase-free TE or water.
• Reverse transcriptase, 200 U/L (SuperScript II; Life Technologies)
  • First strand buffer <R> and DTT, both required for preparation of the master reagent mix in Step 3, are provided with SuperScript II.
• Total RNA (from Protocol 3 or 4)
• Unlabeled dNTPs (low dTTP) stock <R>

• Heat block set at 65C
• MinElute kit (QIAGEN, catalog no. 28004)
• Thermal cycler

• 1. Prepare the following RNA/oligo reaction mixtures in separate microcentrifuge tubes.
• 2. Heat the tubes for 10 min to 65C, and cool on ice to anneal the primers to the RNA.
• 3. While the RNA/oligo reaction mixtures are incubating, prepare two master reaction mixtures, one containing Cy3-dUTP and one with Cy5-dUTP, in a volume sufficient to transfer 14.6 L of each to an RNA/oligo reaction mixture.
• 4. To the Cy3 RNA/oligo reaction mixture, add 14.6 L of the Cy3 master reaction mixture (Step 3) to each tube. To the Cy5 RNA/oligo reaction mixture, add 14.6 L of the Cy5 master reaction mixture (Step 3) to each tube. Incubate for 1 h at 42C.
• 5. Add 1 L of SuperScript II enzyme (200 U/L) to each reaction and thoroughly mix the reaction components with a pipette. Incubate for an additional 1 h.
• 6. Degrade the RNA by adding 15 L of 0.1 N NaOH to each reaction and incubating for 10 min at 65C70C.
• 7. Neutralize each reaction by adding 15 L of 0.1 N HCl.

If you want to determine the amount of fluorophore incorporated, then clean up the Cy5- and Cy3-labeled cDNA samples on separate MinElute columns. However, if thin coverslips and a small probe volume will be used during the hybridization (Protocol 10), it may be necessary to purify the Cy5- and Cy3-labeled cDNA samples together or use vacuum centrifugation to reduce the volume after elution.

• 8. Add 600 L of Buffer PB (binding buffer) to each sample.
• 9. Assemble a MinElute column on a 2-mL collection tube.
• 10. Add the entire 660 L or 720 L (volume depends on whether each color sample is being cleaned up separately or together) to a MinElute column. Centrifuge the column for 1 min at 10,000g. Discard the flowthrough, and reuse the 2-mL tube.
• 11. Add 750 L of Wash buffer PE to the column. Centrifuge for 1 min at 10,000g. Discard the flowthrough, and reuse the 2-mL tube.
• 12. Centrifuge again at maximum speed for 1 min to remove residual ethanol.
• 13. Place the column in a fresh 1.5-mL tube. Add 10 L of H₂O to elute. Allow the Elution buffer to stand for at least 2 min.
• 14. Centrifuge at maximum speed for 1 min. Add 10 L of H₂O to elute. Allow the Elution buffer to stand for at least 2 min before spinning.
• 15. Centrifuge at maximum speed for 1 min.
• 16. Measure the volume of the eluate for each sample. The volume should be 18 L for each column.

• aa-dUTP mixture (50) <R>
• Anchored oligo(dT) (Life Technologies)
  • Anchored oligo(dT) primer is a mix of 12 primers, each having a sequence of 20 dT residues followed by two nucleotides VN, where V is dA, dC, or dG and N is dA, dC, dG, or dT. Thus, the VN anchor restricts annealing of the primer to the 5 end of the poly(A) tail of mRNA.
• Cyanine dyes (typically Cy5 and Cy3) (GE Healthcare Life Sciences)
  • Dye comes dried and should be resuspended in 11 L of DMSO. This amount of material is sufficient for three labeling reactions. If the entire amount will not be used, prepare 3-L aliquots, dry down in a rotary vacuum, and store them at 20C.
• DTT (0.1 M)
• EDTA (0.5 M)
• HEPES (1 M, pH 7.5)
• NaHCO₃ (50 mM, pH 9.0)
• NaOH (1 N)
• Reverse transcriptase (SuperScript II; Life Technologies)
• Reverse transcriptase buffer (5) (Life Technologies)
• RNasin (ribonuclease inhibitor)
• Total RNA (from Protocol 3 or 4)

• Heat block set at 67C, 70C, and 95C
• Lightproof box
• MinElute kit (QIAGEN, catalog no. 28204)
• Thermal cycler (42C)
• Zymo column (Zymo Research, catalog no. D3024)

• 1. Combine 30 g of total RNA and water to a final volume of 14.5 L. Add 1 L of 5 g/L anchored oligo(dT). Mix by pipetting. Heat for 10 min at 70C.
• 2. Cool the tube on ice for 10 min.
• 3. Pulse-centrifuge to bring the condensate to the bottom of the tube.
• 4. Prepare the Master mix just before use (be sure to add the enzymes last):
• 5. Add 14.5 L of Master mix to the tube of RNA. Pipette to mix. Incubate the reaction for 2 h at 42C.
• 6. Incubate the tube for 5 min at 95C. Transfer immediately to ice.
• 7. Add 13 L of 1 N NaOH and 1 L of 0.5 M EDTA to hydrolyze the RNA. Mix the reagents and pulse-centrifuge. Incubate the tube for 15 min at 67C.
• 8. Neutralize the reaction with 50 L of 1 M HEPES (pH 7.5). Vortex the tube and pulse-centrifuge.
• 9. Purify the reaction over a Zymo column to remove unincorporated nucleotides.
  • i. Add 1 mL of Binding buffer to the reaction. Mix by pipetting. Load half of the material onto the column. Centrifuge for 10 sec at maximum speed. Discard the flowthrough.
  • ii. Add the remainder of the reaction. Centrifuge for 10 sec at maximum speed. Discard the flowthrough.
  • iii. Add 200 L of Wash buffer to the column. Centrifuge for 30 sec at maximum speed.
    • Typically, multiple individual samples will be labeled simultaneously, with at least two reactions for a given two-color microarray.
  • iv. Add another 200 L of Wash buffer to the column. Centrifuge for 1 min at maximum speed. Discard the flowthrough, and centrifuge for 1 min at maximum speed.
  • v. Add 10 L of 50 mM NaHCO₃ (pH 9.0) to the filter. Incubate for 5 min at room temperature. Centrifuge for 30 sec at maximum speed to elute the cDNA.
• 10. Couple cyanine dyes to the aa-dUTP incorporated cDNAs by adding the eluted material directly to 3 L of Cy5 or Cy3 dye resuspended in DMSO. Incubate for 1 h to overnight at room temperature in a lightproof box or drawer.

• 11. Purify the cDNA using a MinElute kit, following the manufacturer's instructions.

• Cy3-dCTP and Cy5-dCTP (GE Life Sciences)
• dNTP mixture (10) <R>
• EDTA (0.5 M, pH 8.0)
• Genomic DNA (or from Protocol 2)
• Human C_ot-1 DNA (1 g/L) (GIBCO)
• Klenow fragment of E. coli DNA polymerase I (4050 units/L)
• Poly(dA-dT) (Sigma-Aldrich)
  • Make a 5 g/L stock in nuclease-free water. Store the stock at 20C.
• Random primer/buffer solution <R>
  • This can be obtained from Life Technologies BioPrime labeling kit or can be made in the laboratory.
• TE (pH 7.4)
• Yeast tRNA (GIBCO)
  • Make a 5 g/L stock in nuclease-free water.

• Heat block set at 37C and 95C100C
• Microcon 30 spin column (Millipore)

• 7. Combine the Cy3- and Cy5-labeled samples, and add 450 L of TE (pH 7.4).
• 8. Add the combined samples to a Microcon 30 spin column. Centrifuge the spin column for 1011 min at 10,000g in a microcentrifuge.
• 9. Discard the flowthrough.
• 10. If hybridizing to homemade microarrays, add blocking nucleotides at this step:
  • If using a commercial microarray, refer to the manufacturer's protocols for specific hybridization buffers and blocking nucleotides.
• 11. Add another 450 L of TE, and centrifuge as in Step 8.
• 12. Measure the volume of the liquid remaining in the column. If necessary, centrifuge for 1 min. The desired probe volume is 20 L. The exact volume will depend on the size of the coverslip that contains the microarray. For small microarrays this volume may be as low as 12 L (see Protocol 10, Step 3).
• 13. Discard the flowthrough. Recover the labeled DNA from the filter by inverting the column onto a fresh collection tube. Centrifuge for 1 min at 10,000g.
• 14. Proceed to hybridization with the microarray (Protocols 9 and 10).

• aa-dUTP/dNTP mixture (3 mM)
  • Combine 6 L of 50 stock <R> used for cDNA synthesis and 44 L of dH₂O.
• Cyanine dyes (typically Cy5 and Cy3) (GE Healthcare Life Sciences)
  • Dyes come dried and should be resuspended in 11 L of DMSO. This amount of material is sufficient for three labeling reactions. If the entire amount will not be used, prepare 3-L aliquots, dry down in a rotary vacuum, and store them at 20C.
• EDTA (0.5 M, pH 8.0)
• Genomic DNA
  • Prepare genomic DNA according to your favorite protocol. Because the DNA should be fairly pure, avoid using quick and dirty methods. Commercial gDNA isolation kits are suitable, as in Chapter 1, Protocols 12 and 13. As a rule of thumb, the OD_260/280 ratio should be at least 1.8.
• Klenow buffer
• Klenow fragment of DNA polymerase I
• NaHCO₃ (50 mM, pH 9.0)
• Random hexamer
  • N6 random hexamer can be ordered from any oligonucleotide company, made up at 5 g/L in RNase-free TE/H₂O.

• Heat block set at 37C and 100C
• Lightproof box (see Step 11)
• MinElute kit (QIAGEN)
• Zymo columns (Zymo Research, catalog no. D3024)

• 1. For each array, combine 4 g of genomic DNA, 10 g of random hexamer (N6), and dH₂O to a final volume of 42 L. Incubate for 5 min at 100C.
• 2. Cool the tube(s) quickly on ice for 10 min.
• 3. Pulse-centrifuge to bring the condensate to the bottom of the tube.
• 4. On ice, add:
  Pipette to mix. Incubate for 2 h at 37C.
• 5. Add 5 L of 0.5 M EDTA (pH 8.0) to stop the reaction.

• 6. Add 1 mL of Binding buffer to the reaction. Mix well, and load half (527 L) of the reaction mixture onto each of two Zymo columns. Centrifuge the columns for 10 sec at maximum speed.
  • The large volume of Binding buffer is necessary to precipitate single-stranded DNA.
• 7. Discard the flowthrough. Wash the column with 200 L of Wash buffer. Centrifuge for 1 min at maximum speed.
• 8. Repeat Step 7.
• 9. Add 10 L of 50 mM NaHCO₃ (pH 9.0) to each column filter. Incubate for 5 min at room temperature.
• 10. Elute the DNA from the column by centrifuging for 1 min at maximum speed.
• 11. Couple cyanine dyes to the aa-dUTP-incorporated cDNAs by adding the eluted material directly to 3 L of Cy5 dye or Cy3 dye resuspended in DMSO. Incubate for 1 h to overnight at room temperature in a lightproof box or a drawer.

• 12. Purify the cDNA using a MinElute kit, following the manufacturer's instructions.

• Ethanol (95) (see Step 11)
• 1-Methyl-2-pyrrolidinone
  • Use only HPLC grade. If it has become slightly yellowed, then it is no longer usable, having absorbed excess water.
• Microarrays printed onto glass slides (either homemade [Protocol 1] or purchased)
• Sodium borate (1 M, pH 8.0)
• SSC (0.5) <A>
• Succinic anhydride
  • The stock bottle of solid succinic anhydride should be stored under desiccation and vacuum (or under nitrogen).
    Do not use if exposed to moisture!

• Beakers (500 mL and 4 L)
• Centrifuge, fitted with microtiter plate carrier
• Glass-etching pen
• Glass slide racks and wash chambers (e.g., Thermo Scientific Fisher, catalog no. NC9516192)
• Gloves, powder-free
• Heat block set at 80C (see Step 5)
• Heating plate set at 80C
• Humid chamber, for standard-size glass slides (e.g., Sigma-Aldrich, catalog no. H6644)
• Microarray slide box, plastic
• Orbital shaker
• Stir bar that fits a 500-mL beaker
• Water bath set at 80C

• 3. Fill the bottom of a humidifying chamber with 0.5 SSC. Suspend the arrays face-up over the 0.5 SSC, and cover the chamber with a lid.
• 4. Rehydrate until all of the microarray spots glisten (usually 15 min at room temperature).
  • Allow the spots to swell slightly, but do not let them run into each other. Insufficient hydration results in less DNA bound within a spot, and too much hydration will cause spots to run together.
• 5. Dry each array by placing each slide, with the DNA side facing up, for 3 sec on an inverted heat block set at 80C.
• 6. Place the arrays in a slide rack. Place the slide rack into an empty slide chamber that is sitting on an orbital shaker.

• 7. Prepare the blocking solution by measuring 335 mL of 1-methyl-2-pyrrolidinone into a clean, dry 500-mL beaker. Dissolve 5.5 g of succinic anhydride using a stir bar.
• 8. Immediately after the succinic anhydride dissolves, mix in 15 mL of 1 M sodium borate (pH 8.0). Quickly pour the buffered blocking solution into a clean, dry glass slide dish.
• 9. Plunge the slides rapidly into the blocking solution, and vigorously shake the slide rack manually, keeping the slides submerged at all times. After 30 sec, place a lid on the glass box, and shake gently on the orbital shaker for 15 min.
• 10. Drain excess blocking solution from the slides for 5 sec. Submerge the slide rack into an 80C water bath. Gently swish the rack back and forth under the water for a few seconds. Incubate for 60 sec.
• 11. Quickly transfer the rack to a glass dish of 95 ethanol, and plunge to mix.
  • Make sure that the ethanol is crystal clear. Do not use if it contains particulates or is cloudy.
• 12. Take the entire glass dish with slide rack still submerged to a bench-top centrifuge. (Be sure to have an equivalently loaded slide rack ready to serve as a balance in the centrifuge.) Drain excess ethanol off the slides for 5 sec. Quickly, place the slide rack onto a microtiter plate carrier, and centrifuge in a bench-top centrifuge for 3 min at 550 rpm.
• 13. After centrifugation, the slides should be clean and dry. Remove the slides from the rack and store them in a plastic microscope slide box. Arrays may be used immediately, or can be stored for at least 34 mo in a desiccator at room temperature.

• C_ot-1 DNA (GIBCO)
  • C_ot-1 DNA is available commercially at 1 g/L. Before hybridization, concentrate C_ot-1 DNA from 1 g/L to 10 g/L in a rotary vacuum device.
• Cy5- and Cy3-labeled nucleic acids (from Protocol 5, 6, 7, or 8)
• HEPES (1 M, pH 7.0)
• Microarrays printed onto glass slides (either homemade [Protocol 1] or purchased)
  • If homemade, slides must be blocked as per Protocol 9 before hybridization.
• Poly(A) RNA (10 g/L) (Sigma-Aldrich, catalog no. P9403)
• SDS (10)
• SSC (20) <A>
• Yeast tRNA (10 g/L) (GIBCO, catalog no. 15401-011)

• Coverslips
  • Use either 2260 regular thin coverslips or 2260 Erie M-series lifter slips.
• Dishes for washing microarrays (see Step 13)
• Gloves, powder-free
• Heat block set at 95C100C
• Hybridization chambers
• Lightproof box
• Microarray scanner
  • When using homemade microarrays, the standard scanner is the GenePix 4000B (Molecular Devices).
• Microarray slide box
• Paper towels
• Water bath set at 65C

If you have already combined the Cy dyes and added blocking material and buffer, skip to Step 4.

• 1. Combine the following blocking nucleic acids:
• 2. Combine Cy5- and Cy3-labeled nucleic acids.
• 3. Prepare the complete hybridization solution as shown in the table below. To avoid introducing bubbles into the solution, do not vortex after adding SDS.
• 4. Denature the probe by heating the hybridization solution in a water-filled heat block for 2 min at 95C100C.
• 5. Let the probe sit for 10 min in the dark at room temperature.
• 6. While the probe is sitting at room temperature, set up the necessary number of hybridization chambers. Open each chamber on a clean, flat surface, and place a microarray slide into a chamber with the array side facing up.
• 7. Centrifuge the solution at 14,000 rpm for 5 min at room temperature.

• 8. Carefully pipette the probe solution as a single drop onto one end of the microarray surface. Avoid creating any bubbles during pipetting, and be careful not to touch the microarray surface with the pipette tip (Fig. 1).
  • Leave 2 L of probe behind in the tube. If the probe precipitates upon application to the slide, see Troubleshooting.
• 9. Carefully apply a coverslip by placing one edge of the coverslip on the slide near the probe and slowly lowering the other edge, using another coverslip as a lever and wedge to lower it (Fig. 1).
• 10. Close the chamber, and immediately submerge it in a 65C water bath.
  • Be careful not to tilt the chamber. Metal tongs may be used to place the hybridization chamber into the water bath.
• 11. If hybridizing multiple arrays, repeat Steps 810.
  • Be quick and efficient because the probes should not sit at room temperature for widely varying times. Try to have all the probes onto slides within 1015 min.
• 12. Incubate the chambers for 1620 h at 65C.

• 13. Prepare dishes with the following solutions:
• 14. If there is one, turn on the ozone scrubber connected to the scanner at least 15 min before scanning the arrays.
  • This is particularly useful in summer months when ozone levels are high. Bleaching of Cy5 dye becomes a significant problem in urban environments in the summer. If you choose not to use an ozone scrubber, consider performing hybridizations on cool or rainy days.
• 15. Turn on the scanner, letting it warm up for at least 15 min to allow the laser outputs to stabilize.
• 16. Launch the GenePix Pro software, and let it connect to the scanner and the network hardware key.
• 17. Remove a hybridization chamber from the water bath. Working quickly, dry the chamber with a paper towel, open the chamber, remove the slide, and using your gloved hand, tap the slide against the bottom of the Wash 1A dish until the coverslip gently slides off the slide.
• 18. Transfer the slide to a presubmerged rack in Wash 1B. Keep the slide in Wash 1B until all arrays have been transferred.
• 19. Quickly transfer the rack from Wash 1B to Wash 2 (tilting the rack back and forth a couple of times to remove excess wash solution). Plunge the rack up and down several times in Wash 2. Incubate for 5 min with occasional plunging up and down.
• 20. Quickly transfer the rack from Wash 2 to Wash 3 (tilting the rack back and forth a couple of times to remove excess wash solution). Plunge the rack up and down several times in Wash 3. Incubate for 5 min with occasional plunging up and down.
• 21. As quickly as possible, transfer the rack of slides from Wash 3 to a tabletop centrifuge, with paper towels under the rack, and centrifuge at 500600 rpm for 5 min.
• 22. Place the microarrays in a lightproof box, and begin scanning immediately.
  • Typically, four to five arrays are washed at a time. If more than five were hybridized, then the next set of slides should be washed as the last array from the first batch is being scanned.

• 23. Open the scanner door, and insert a slide with the array features facing down and the barcode/label closest to the edge of the scanner.
• 24. Run a preview scan using the double-arrow button on top of the right-hand toolbar to visualize the array.
  • This performs a low-resolution (40-m) scan so that the location of array features can be visualized.
• 25. After the preview scan is completed, select the Scan Area button in the left-hand Tools group. Use the mouse to draw a rectangle around the region containing features.
  • This limits the data scan to just this area, which reduces the time needed for each scan.
• 26. Click on the Hardware Settings button on the right-hand side of the window to bring up the Settings box.
• 27. Use the Auto Scale Brightness/Contrast button on the left-hand side to adjust the brightness and contrast of the image (both colors will be affected). Switch between the red and green channels by clicking on the respective radio buttons on the top left-hand side of the image tab.
• 28. Set the Pixel Resolution to 10 m, then start the data scan. Zoom into the top half of the scanned image, and by eyeballing the image, adjust the PMT gain settings in both the red and green channels so that the average signal intensities balance out.
  • This takes some practice, although Step 29 describes how to do this with the aid of the intensity histograms. By eye, the goal is to balance the array so that the color yellow dominates, with roughly equal numbers of green and red spots. An array with mostly green spots will need the red channel PMT increased, and vice versa.
  • Normally, the PMT gain for the green channel (532 nm) is around 100 less than the red channel (635 nm). Adjust the settings so that any landing lights (bright spots printed near the top of the array corresponding to highly expressed genes, etc.) and some positive control spots on the array are saturated, but not many of the other features. Saturated pixels are drawn as white on the image.
  • Overall, the goal is to minimize the length of time spent scanning the array, and because some color imbalance can be normalized out later, it is more important to be fast than to be absolutely precise with regard to color balance.
• 29. Alternatively (to the previous step), view the Histogram tab to check on the red/green ratio. Set the Min and Max Intensity fields in the Image Balance area on the left-hand side to 500 and 65,530, respectively. The goal is for the Count Ratio field to be 1.0, although this is not as important as having the two histogram curves as close to overlapping as possible.
• 30. Once the PMT Gains for both channels are balanced, stop the scan by hitting the red Stop Scan button on the right-hand side of the window. Change the pixel resolution in the Hardware Settings window to 5 m, and ensure that the Lines to Average field is at 1. (More will not be detrimental to the scanned image, but the scanning will take much longer.)
• 31. Start the scan again with the new settings. Record the PMT gains for each array in an Excel spreadsheet, along with any special observations/notes for the slide (e.g., the red signal is very dim, etc.). Save the spreadsheet in the same folder as the image files.
• 32. When the entire array is scanned, click the File button on the right-hand side of the window. Select Save Images, and from the Save As Type field, select Single-Image TIFF Files. Make sure that both wavelengths are selected. Name the arrays with the identifier printed on the label, then hit the Save button.
  • There should be two TIFF (.tif) files, one with the data from the green channel (532 nm) and one with the data from the red channel (635 nm).
• 33. Scan the next array, repeating Steps 2332. Balancing the two PMTs must be done for every microarray, as different RNA or DNA samples label with different efficiency.
• 34. After all of the slides have been scanned, quit the GenePix Pro program, and turn off the scanner.

• 35. Open the GenePix software, and open the image file in question.
• 36. Open the .gal file, which describes what DNA is found in each spot. This file will either be provided by the microarray manufacturer or it can be generated using the ArrayList generator found in GenePix.
• 37. Align each pattern block (corresponding to one print pin) over the image so that image spots are roughly aligned with circles in the .gal file.
• 38. Find features for each block by pressing [F5]. GenePix will move .gal file circles to cover the closest spots and will also flag spots where the intensity is too low.
• 39. For each block, scan the entire sector by eye, and manually flag spots that are scratched or appear to be fluorescent dust rather than DNA.
• 40. After all blocks have been flagged, hit the Analyze button to extract the .gpr file.
• 41. At this point, either save the .gpr output file or choose Flag features to automatically impose thresholds.
  • We typically use this to flag all spots with a signal-to-noise ratio in each channel (532, 635) of <3.
• 42. Save the .gpr file.