Microarray Analysis

3.2.1. SNP Genome Scanning

1. Multiplex PCR reactions are performed as 24 separate reactions in 96-well micro-titer plates (Affymetrix mapping assay manual).

2. DNA from individuals or pools is amplified in 24 separate reactions containing 6 ng of DNA from each individual and 84 multiplex PCR primer pairs.

3. Primary PCR products are subjected to labeling PCR as described by Affymetrix. The 24 labeled PCR samples corresponding to each genomic DNA sample are then pooled, diluted in hybridization buffer, heated to 95°C, cooled to 4°C, then hybridized with Affymetrix HuSNP GeneChip microarrays at 45°C for 18 h.

4. Arrays are washed with two changes of 6X SSPE/0.01% Triton at 25°C and then with six changes of 4X SSPE/0.01% Triton at 35°C. DNA hybridized to the washed microarrays is stained using 2 |g/mL phycoerythrin-conjugated streptavidin in 6X SSPE, 0.01% Triton, 0.5 mg/mL BSA at 25°C for 30 min. Stained arrays are washed with four changes of 6X SSPE/0.01% Triton at 25°C and then scanned using a GeneArray Laser Scanner (Hewlett Packard).

Fig. 1. C57BL/6 mice were acutely treated with 20 mg/kg morphine subcutaneous-ly and separate sub groups treated chronically with either the same dose of morphine daily or saline. Mice were sacrificed 4 h after final injections and the brains quickly dissected to extract RNA for CBir gene expression study. The expression of CBir RNA was compared by real-time PCR using the gene-specific probe and primers. Acute and chronic treatment with 20 mg/kg morphine produced a reduction in the expression of CBir gene transcripts in the striatum (str), midbrain (mid), and hippocampus (hip).

Fig. 1. C57BL/6 mice were acutely treated with 20 mg/kg morphine subcutaneous-ly and separate sub groups treated chronically with either the same dose of morphine daily or saline. Mice were sacrificed 4 h after final injections and the brains quickly dissected to extract RNA for CBir gene expression study. The expression of CBir RNA was compared by real-time PCR using the gene-specific probe and primers. Acute and chronic treatment with 20 mg/kg morphine produced a reduction in the expression of CBir gene transcripts in the striatum (str), midbrain (mid), and hippocampus (hip).

3.2.2. SNP Genome Scanning Analysis

With genotyping, individual DNAs are made using Affymetrix GeneChip software. Allellic frequencies in pooled DNA samples are assessed based on data from the "cell" files and an algorithm averaging the hybridization intensity signals from the 8-16 features per chip that provided perfectly complementary hybridization to the alleles termed A or B for each SNP.

1. The background, determined as the average of the lowest 5% of intensity values on each microarray, is subtracted from the fluorescence intensity of each cell of that microarray.

2. Background-corrected values are normalized by dividing each value by the average of the highest 5% of intensity values on each microarray.

3. Normalized hybridization intensities from the microarray elements that corresponded to the perfect match A and perfect match B alleles for each SNP are each averaged.

4. A/B ratios are determined by dividing average normalized A values by average normalized B values.

5. Arctangent transformations are applied to each ratio to improve combination of data from experiments with different absolute intensity values.

6. Arctan A/B ratios for abusers are then divided by arctan A/B ratios for controls to form an abuser/control ratio, in our primary analysis.

7. Means of three replications for each experiment identified SNPs with abuser/control hybridization ratios in the top and bottom 5% of the distribution for the European-American abuser vs control comparisons.

8. Reproducibility of these "candidate-positive" SNPs was sought by asking if they were also in the upper or lower 7.5% of the distribution of hybridizaton ratios in data from two replications in African American abuser vs control samples. Physical locations of these reproducibly positive SNPs and of markers linked to alcohol or nicotine dependence in previous studies were sought in Mapviewer build 22 (5/01) and related NCBI programs by two independent investigators. Additional locations were sought in Mapviewer build 24 (9/01).

9. Allele calls from individual DNA samples are also compared to results from pooled DNAs from the same individuals. Regression analyses compared allele frequencies for SNPs in most of the individuals studied (Fig. 2).

10. Test-retest variability was studied in replicated microarray results from DNA pools prepared in duplicate or triplicate from the same DNA stocks from the same individuals.

11. Results are compared to those expected by chance using Monte Carlo simulations, using the C computer language. For the initial results of SNPs that were positive in both Caucasian and African-American samples, 100,000 Monte Carlo trials were carried out. In each trial, 148 markers (5% + 5% of 1493) were sampled with replacement, and subsequently 222 (7.5% + 7.5% of 1493) were sampled with replacement from the all of the 1493 markers. The number of markers common to both samples were then determined. In only 11 of these trials did we find at least 42 markers in common (p = 0.00011).

12. To model the chance probabilities of obtaining positive markers as close to each other as the three closest pairs identified here, we used 1 million simulation trials in which 42 markers were randomly positioned on a 3.2 billion bp genome and pairwise distances between each marker pair were determined.

13. We assessed the number of simulations in which the smallest distance was <0.7 Mbp, the second smallest distance was <1.5 Mbp, and the third smallest distance was <2.2 Mbp (Mapviewer build 22 distances modeled corresponding to build 24 distances are 0.2, 2.8, and 6.6 Mb).

14. To model the chance probabilities of obtaining positive markers as close to markers previously reported to be linked to ethanol or nicotine addiction, we used more than 100 million simulation trials in which 61 markers were randomly positioned on a 3.2 billion bp genome, then 31 markers randomly placed on the same genome and pairwise distances between each marker pair, consisting of one member of the first set and one member of the second set, were determined.

15. We assessed the number of simulations in which at least seven of the distances were <1 Mbp (Mapviewer 22 distances modeled; corresponding build 24 distances are 0, 0.1,0.4,0.4,0.4,2.3, and 2.7 Mb). Linkage disequilibrium was assessed for data from individual genotypes determined for 20 control subjects using Arlequin.

Individual SNP Markers

Pooled SNP Markers

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Homozygous A

Homozygous B

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Heterozygous A&B

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Fig. 2. Hybridization of Affymetrix HuSNP GenChips using biotinylated PCR probes synthesized from individual or pooled DNA samples. A SNP marker is represented by four oligomer "quartets" in which two perfect matches (PM) correspond to allele A and B, respectively, and two mismatches (MM) correspond to homomeric bases of the respective SNP. As shown in the individual SNP markers and pooled SNP markers, individual genotypes could be called according intensities of allele A, B, or AB. Pooled allele fre- c;

quencies could be derived from intensity ratio of allele A and B using arctangent transformation. £

3.2.3. Gene Expression Array

1. Male mice (age: 14-16 wk and weight: 20-30 g) are kept under 12-h light/dark cycle with free access to water and food. A slow-releasing morphine pellet (25 mg) or placebo pellet (25 mg) (NIDA, Rockville, MD) was implanted subcutaneously in each mouse under isofluorene anesthesia. Mice with morphine pellets developed withdrawal syndrome after 4 d. On d 5, mice were sacrificed by cervical dislocation. Mu knockout mice and wild-type littermates in the experiments were described previously. The mouse brain was dissected rapidly and frozen immediately in liquid nitrogen.

2. Total RNA was extracted from mouse whole brain using RNAzolB (Tel-Test, Inc. Friendwood, TX) protocol. mRNA was isolated from total RNA using Straight A'sTM System (Novagen, Madison, WI) protocol.

3. Double-stranded cDNA was synthesized using SuperScript™ Choice System protocol (GIBCO/BRL, Rockville, MD) with (dT)24-T7 primer. In order to amplify cDNA linearly, cRNA probes were transcribed in vitro in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmindale, NY) using MEGAscript T7 protocol (Ambion, Auastin, TX). Biotin-labeled cRNA was purified with RNeasy Mini Kit (Qiagen, Valencia, CA) and fragmented with alkaline to 100-300 bases before hybridization.

4. Mu6500 GeneChips (Affymetrix, Santa Clara, CA) are hybridized with biotin-cRNA overnight at 45°C according to the manufacture's protocol and stained with R-phycoerythrin conjugated streptavidin (Molecular Probes, Eugene, OR). After wash, Mu6500 chips were scanned with the Hewlett Packard GeneArray Scanner.

5. The expression levels of various genes are analyzed with Affimetrix Microarray Suite 5.0 software. The experiments for chronic morphine treatment and mu knockout mice are repeated independently three times with the Mu6500 GeneChip.

6. The data from Affymetrix analysis were transferred into Microsoft Access database, and the reproducible gene expression changes were extracted by linking data from separate experiments.

7. Hybridization of Affymetrix Mu6500 GenChip using biotinylated cRNA probes synthesized from brain cDNAs of dopamine (DAT) and serotonin (SET) transporter gene knockout mice or double (DAT/SET) knockout mice is shown in Fig. 3. A gene transcript is represented by matches of 11-16 oligomers. The hybridization intensities of the perfect matches from separate knockout mouse brains are compared as shown.

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