Tiling Arrays

  • Human tiling arrays are designed by dividing the non-repetitive sequence of a genomic region or regions into contiguous 35 base pair units. A 25-mer is designed from each of these units (Promoter and Whole Genome arrays) or overlapping 7 base pair (ENCODE array) or 16 base pair (Chromosome 21/22 array) units. The collection of these 25-mers comprises a tiling array. The arrays are useful for the localization of the genomic binding sites of transcription factors and other chromatin associated proteins. Experimental turn-around times are usually four to five business days.

    The Affymetrix GeneChip® Chromatin Immunoprecipitation (ChIP) Assay is designed to generate double-stranded labeled DNA targets that identify sites of protein-DNA interactions or chromatin modifications on a genome-wide scale. This assay has been designed specifically for use with Affymetrix GeneChip Tiling Arrays for ChIP-on-chip studies in order to study transcription factor binding sites, histone protein modifications, and other chromatin-protein interactions. The ChIP experiments can be used as a powerful tool to complement RNA transcription studies because they enable researchers to study the DNA-protein interactions that regulate gene expression. Following the protocol, cells are first fixed with formaldehyde to crosslink DNA to any associated proteins. The cells are then lysed and DNA is sheared into smaller fragments using sonication. Protein-DNA complexes are then immunoprecipitated with an antibody directed against the specific protein of interest. Following the immunoprecipitation, crosslinking is reversed, samples are protease-treated, and the purified DNA sample is amplified using a random-primed PCR method. Subsequently, targets are fragmented and labeled to hybridize onto GeneChip Tiling Arrays. By comparing the hybridization signals generated by an immunoprecipitated sample versus an antibody-negative or non-specific antibody control, the regions of chromatin-protein interaction can be identified. Due to the variability and the length of time needed to complete the upstream part of this reaction, usually the investigating PI will perform the immunoprecipitation reaction and provide the core with the proteinase-treated immunoprecipitated DNA that has been purified.

    The Affymetrix GeneChip Whole Transcript (WT) Double-Stranded Target Assay is designed to be used in conjunction with Affymetrix GeneChip Tiling Arrays for unbiased genome-wide transcript mapping studies. Both the amplified and unamplified protocols produce double-stranded, labeled DNA targets that interrogate the genome for all regions of expression. For users of single tiling array designs, such as model organism tiling arrays, the protocol without an amplification step is recommended and has the advantage of quicker target preparation time when sample enrichment is not required. The amplified protocol requires at least 3μg of total RNA or 300ng of poly-A+ RNA as starting material. The RiboMinus™ Transcriptome Isolation kit from Invitrogen is used to remove the 28S and 18S ribosomal RNAs from total RNA samples, thereby maximizing sensitivity and specificity of hybridizations. In contrast, the rRNA reduction is not needed for the poly-A+ RNA. Starting from at least 3μg total RNA, the amount of labeled target produced is sufficient to hybridize up to nine arrays, allowing for re-hybridizations of saved hybridization cocktails. However, the unamplified protocol requires 7μg of total RNA as starting material and has been designed specifically for the use with single-array design tiling arrays for RNA mapping applications in order to probe the genome for all regions of expression. Random-prime cDNA is generated from total RNA without enrichment or amplification of the mRNA pool. Following the recommended protocol, the amount of labeled target produced by this procedure is sufficient to hybridize to a single array.

    Using the amplified target assay, the total RNA is first purified and enriched by rRNA reduction. The newly isolated mRNA is copied into double-stranded (ds) DNA using the GeneChip WT Amplified Double-Stranded cDNA Synthesis kit and amplified by in vitro transcription into cRNA using the same kit. The cRNA is split into three subsequent second cycle double-stranded cDNA synthesis reactions, which incorporate deoxyuridine into each cDNA strand at predefined ratios. The dU-incorporated dsDNA is fragmented using apurinic/apyrimidinic endonuclease (APE 1) and uracil DNA glycosylase (UDG) enzymes and then labeled using terminal deoxynucleotidyl transferase (TdT) and Affymetrix proprietary DNA Labeling Reagent supplied in the GeneChip WT Double-Stranded DNA Terminal Labeling kit. The labeled double-stranded target is then ready to add to the hybridization cocktail and hybridize onto GeneChip Tiling Arrays. However, the unamplified target assay forgoes the rRNA reduction step, creates cDNA, and fragments and labels this target for hybridization using the same kits.

    Quantity
    Enough cells need to be grown for the number of immunoprecipitation (IP) reactions to be performed (usually 5x107 cells per IP for suspension cells, depending on IP efficiency). Prepare enough cells for two IP reactions. An antibody-minus (Ab- or mock IP) or nonspecific IgG is recommended as a negative control using the same number of cells as the IP condition. The Ab- target would be treated identically to the experimental sample to serve as the “control” group in the downstream two-sample analysis. Approximately 0.5 to 2x108 cells per IP are used. For example, you should grow 200mL of 1x106cells/mL for a total of 2x108 cells. The analysis begins with purified immunoprecipitated DNA and negative antibody controls that are provided by an investigator. The ChIP protocol requires 10µL of purified DNA or negative antibody control to amplify these targets. Please provide the entire elution amount of your targets.

    The amplified protocol requires at least 3μg of total RNA as starting material, where the concentration should not fall below 0.24μg/μL. In other words, a minimum of 3μg of total RNA should be suspended in a maximum of 12.5μL of solution in volume. We recommend samples concentrated at 1μg/µL for simplicity. Please provide an extra 2ug of total RNA in addition to the experimental quantity submitted in order to account for the protocol checkpoints and any concentration variations, should your sample concentrations be off.

    The unamplified protocol requires 300ng of poly-A+ RNA as starting material, which should be suspended in a maximum of 4μL of solution in volume. We recommend samples concentrated at 100ng/µL for simplicity. Please provide an extra 200ng of poly-A+ RNA in addition to the experimental quantity submitted in order to account for the protocol checkpoints and any concentration variations, should your sample concentrations be off.