Biomedical subjects

Lue Ping Zhao

Publications and source records attributed to Lue Ping Zhao.

At least 19 recordsLinked to original sources

PubMed2007

A haplotype-linkage analysis method for estimating recombination rates using dense SNP trio data.

Meiotic recombination is an important evolution force in shaping human genomes and creating human diversity. Recent explorations suggest that meiotic recombination events tend to happen in certain regions of the genome, leading to the hypothesis of recombination hot spots. To identify such hot spots, we describe an empirical method for estimating the recombination rate, which reflects both historical and current meiotic events, using unphased genotypes from nuclear families, in particular, parents-child trios. The key idea is that use of the haplotypic polymorphisms with multiple adjacent SNPs will increase the number of informative meioses and hence would improve the power of linkage analysis. Since haplotypes of individuals are not directly observed, e.g., in HapMap data, we infer the haplotypes simultaneously while estimating recombination rate. We refer this described method as haplotype-linkage (HALIN) method. Our simulation results show that HALIN gives unbiased estimates of recombination rate. We apply HALIN to analyze the genotype data of chromosome 20 from HapMap data and compare the results to the results using two existing methods, Bayesian coalescent method described by McVean et al. [2004] science 304:581-584 and empirical method by Clarke and Cardon ([2005] Genetics). Results suggest that HALIN identified 75 hot spots on chromosome 20, and 85% of them are also identified by the method by McVean et al. ([2004] science 304:581-584), in addition to a few new hot spots. In comparison with Clarke and Cardon's result, estimated recombination rates (RRs) under these 75 hot spots are significantly greater than those outside of hot spots, which support the general consistency in discovering recombination hot spots between two methods.

Child↗

PubMed2006

Quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human lymphoblastoid TK6 cells dosed with N-hydroxy-4-acetylaminobiphenyl and their relationship to mutation, toxicity, and gene expression profiling.

Gene expression profiles that are anchored to phenotypic endpoints may lead to the identification of signatures that predict mutagenicity or carcinogenicity. The study presented here describes the analysis of DNA adducts in the human TK6 lymphoblastoid cell line after exposure to N-hydroxy-4-aminobiphenyl, a mutagenic metabolite of 4-aminobiphenyl. A validated nano-LC microelectrospray mass spectrometry assay is reported for the detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the principal DNA adduct of 4-aminobiphenyl. Limits of quantification, based on a signal-to-noise ratio of 10:1, are determined to correspond to approximately 27 fg of dG-C8-ABP injected on-column. The assay has been used to measure the steady-state levels of the adduct in the human TK6 lymphoblastoid cell line as a function of dose (0.5, 1.0, and 10.0 microM) and time (2, 6, and 27 h) after exposure to N-hydroxy-4-aminobiphenyl. The levels of dG-C8-ABP adducts in the cells, ranging from 18 to 500 adducts in 10(9) nucleotides, were then correlated to cell toxicity, induced mutation at the TK (thymidine kinase) and HPRT loci, and gene expression profiling through microarray analysis. Cell cultures were evaluated for toxicity by growth curve extrapolation, mutation assays were performed on the HPRT and TK loci, and gene expression profiles were generated by analyses using microarray technology. In the mutation assay analysis, as the toxicant concentration increased, there was an increase in mutation fraction, indicating a direct correlation to metabolite dosing level and mutations occurring at these two loci. Statistical analysis of the gene expression data determined that a total of 2250 genes exhibited statistically significant changes in expression after treatment with N-OH-AABP (P < 0.05). Among the genes identified, 2245 were up-regulated, whereas 5 genes that had functions in cell survival and cell growth and, hence, could be indicators of toxicity, were down-regulated relative to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic markers, such as DNA adduct levels, toxicity, and mutagenicity.

Aminobiphenyl Compounds↗

PubMed2006

A polymorphism in Toll-interleukin 1 receptor domain containing adaptor protein is associated with susceptibility to meningeal tuberculosis.

BACKGROUND: Although meningitis is the most severe form of infection caused by Mycobacterium tuberculosis, the immunopathogenesis of this disease is poorly understood. We tested the hypothesis that polymorphisms in Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), an adaptor protein that mediates signals from Toll-like receptors activated by mycobacteria, are associated with susceptibility to tuberculosis (TB). METHODS: We used a case-population study design in Vietnam with cord-blood control samples (n = 392) and case patients (n = 358) who had either pulmonary (n = 183) or meningeal (n = 175) TB. RESULTS: The TIRAP single-nucleotide polymorphism (SNP) C558T was associated with increased susceptibility to TB, with a 558T allele frequency of 0.035 in control samples versus 0.074 in case patients (odds ratio [OR], 2.25; P < .001). Subgroup analysis revealed that SNP 558T was more strongly associated with susceptibility to meningeal TB (OR, 3.02; P < .001) than to pulmonary TB (OR, 1.55; P = .22). In comparison to the 558CC genotype, the 558TT genotype was associated with decreased whole-blood interleukin-6 production, which suggests that TIRAP influences disease susceptibility by modulating the inflammatory response. CONCLUSIONS: These results provide the first evidence of an association of a TIRAP SNP with the risk of any disease and also suggest that the Toll-like receptor pathway influences susceptibility to meningeal and pulmonary TB by different immune mechanisms.

Adult↗

PubMed2006

A statistical method for predicting splice variants between two groups of samples using GeneChip expression array data.

BACKGROUND: Alternative splicing of pre-messenger RNA results in RNA variants with combinations of selected exons. It is one of the essential biological functions and regulatory components in higher eukaryotic cells. Some of these variants are detectable with the Affymetrix GeneChip that uses multiple oligonucleotide probes (i.e. probe set), since the target sequences for the multiple probes are adjacent within each gene. Hybridization intensity from a probe correlates with abundance of the corresponding transcript. Although the multiple-probe feature in the current GeneChip was designed to assess expression values of individual genes, it also measures transcriptional abundance for a sub-region of a gene sequence. This additional capacity motivated us to develop a method to predict alternative splicing, taking advance of extensive repositories of GeneChip gene expression array data. RESULTS: We developed a two-step approach to predict alternative splicing from GeneChip data. First, we clustered the probes from a probe set into pseudo-exons based on similarity of probe intensities and physical adjacency. A pseudo-exon is defined as a sequence in the gene within which multiple probes have comparable probe intensity values. Second, for each pseudo-exon, we assessed the statistical significance of the difference in probe intensity between two groups of samples. Differentially expressed pseudo-exons are predicted to be alternatively spliced. We applied our method to empirical data generated from GeneChip Hu6800 arrays, which include 7129 probe sets and twenty probes per probe set. The dataset consists of sixty-nine medulloblastoma (27 metastatic and 42 non-metastatic) samples and four cerebellum samples as normal controls. We predicted that 577 genes would be alternatively spliced when we compared normal cerebellum samples to medulloblastomas, and predicted that thirteen genes would be alternatively spliced when we compared metastatic medulloblastomas to non-metastatic ones. We checked the consistency of some of our findings with information in UCSC Human Genome Browser. CONCLUSION: The two-step approach described in this paper is capable of predicting some alternative splicing from multiple oligonucleotide-based gene expression array data with GeneChip technology. Our method employs the extensive repositories of gene expression array data available and generates alternative splicing hypotheses, which can be further validated by experimental studies.

Algorithms↗

PubMed2006

HLA-E, HLA-F, and HLA-G polymorphism: genomic sequence defines haplotype structure and variation spanning the nonclassical class I genes.

Despite several studies that defined the polymorphism of the nonclassical human leukocyte antigen-E (HLA-E), HLA-F, and HLA-G genes, most polymorphisms thus far examined in correlative studies were derived from the coding sequences of these genes. In addition, some discrepancies and ambiguities in the available data have persisted in current databases. To expand the data available and to resolve some of the discrepant data, we have defined protocols that allow for the amplification of 6 to 7 kb of contiguous genomic sequence for each gene, including all of the coding and intron sequences, approximately 2 kb of 5' flanking promoter sequence, and 1 kb of 3' flanking sequence. Using long-range polymerase chain reaction (PCR) protocols, generating either one or two PCR products depending on the locus, amplified genomic DNA was directly sequenced to completion using a set of about 30 primers over each locus to yield contiguous sequence data from both strands. Using this approach, we sequenced 33 genomic DNAs, from Asian, African American, and Caucasian samples. The results of this analysis confirmed several previously reported coding sequence variants, identified several new allelic variants, and also defined extensive variation in intron and flanking sequences. It was possible to construct haplotype maps and to identify tagging single nucleotide polymorphisms that can be used to detect the composite variation spanning all three genes.

Alleles↗

PubMed2006

A fine-scale linkage-disequilibrium measure based on length of haplotype sharing.

High-throughput genotyping technologies for SNPs have enabled the recent completion of the International HapMap Project (phase I), which has stimulated much interest in studying genomewide linkage-disequilibrium (LD) patterns. Conventional LD measures, such as D' and r(2), are two-point measurements, and their relationship with physical distance is highly noisy. We propose a new LD measure, Delta , defined in terms of the correlation coefficient for shared haplotype lengths around two loci, thereby borrowing information from multiple loci. A U-statistic-based estimator of Delta , which takes into consideration the dependence structure of the observed data, is developed and compared with an estimator based on the usual empirical correlation coefficient. Furthermore, we propose methods for inferring LD-decay rates and recombination hotspots on the basis of Delta . The results from coalescent-simulation studies and analysis of HapMap SNP data demonstrate that the proposed estimators of Delta are superior to the two most popular conventional LD measures, in terms of their close relationship with physical distance and recombination rate, their small variability, and their strong robustness to marker-allele frequencies. These merits may offer new opportunities for mapping complex disease genes and for investigating recombination mechanisms on the basis of better-quantified LD.

Alleles↗

PubMed2006

Toward understanding MHC disease associations: partial resequencing of 46 distinct HLA haplotypes.

We carried out a resequencing project that examined 552 kb of sequence from each of 46 individual HLA haplotypes representing a diversity of HLA allele types, generating nearly 27 Mb of fully phased genomic sequence. Haplotype blocks were defined extending from telomeric of HLA-F to centromeric of HLA-DP including in total 5186 MHC SNPs. To investigate basic questions about the evolutionary origin of common HLA haplotypes, and to obtain an estimate of rare variation in the MHC, we similarly examined two additional sets of samples. In 19 independent HLA-A1, B8, DR3 chromosomes, the most common HLA haplotype in Northern European Caucasians, variation was found at 11 SNP positions in the 3600-kb region from HLA-A to DR. Partial resequencing of 282 individuals in the gene-dense class III region identified significant variability beyond what could have been detected by linkage to common SNPs.

Cell Line, Transformed↗

PubMed2006

Genetic association of the antiviral restriction factor TRIM5alpha with human immunodeficiency virus type 1 infection.

The innate antiviral factor TRIM5alpha restricts the replication of some retroviruses through its interaction with the viral capsid protein, leading to abortive infection. While overexpression of human TRIM5alpha results in modest restriction of human immunodeficiency virus type 1 (HIV-1), this inhibition is insufficient to block productive infection of human cells. We hypothesized that polymorphisms within TRIM5 may result in increased restriction of HIV-1 infection. We sequenced the TRIM5 gene (excluding exon 5) and the 4.8-kb 5' putative regulatory region in genomic DNA from 110 HIV-1-infected subjects and 96 exposed seronegative persons, along with targeted gene sequencing in a further 30 HIV-1-infected individuals. Forty-eight single nucleotide polymorphisms (SNPs), including 20 with allele frequencies of >1.0%, were identified. Among these were two synonymous and eight nonsynonymous coding polymorphisms. We observed no association between TRIM5 polymorphism in HIV-1-infected subjects and their set-point viral load after acute infection, although one TRIM5 haplotype was weakly associated with more rapid CD4(+) T-cell loss. Importantly, a TRIM5 haplotype containing the nonsynonymous SNP R136Q showed increased frequency among HIV-1-infected subjects relative to exposed seronegative persons, with an odds ratio of 5.49 (95% confidence interval = 1.83 to 16.45; P = 0.002). Nonetheless, we observed no effect of individual TRIM5alpha nonsynonymous mutations on the in vitro HIV-1 susceptibility of CD4(+) T cells. Therefore, any effect of TRIM5alpha polymorphism on HIV-1 infection in primary lymphocytes may depend on combinations of SNPs or on DNA sequences in linkage disequilibrium with the TRIM5alpha coding sequence.

Adult↗

PubMed2006

Use of gene expression microarrays for the study of acute leukemia.

Genetic lesions found in acute leukemia drive the pathology of the disease in addition to forming reliable classifications of prognosis. However, there is still a reasonable heterogeneity of response among cases with the same genetic lesion. Moreover, many leukemia cases have no detectable genetic marker and these cases have marked heterogeneity of response. How can we learn more about the genes and pathways involved with leukemogenesis and response in the midst of such complexity? Gene expression microarrays are experimental platforms that allow for the simultaneous evaluation of the thousands of mRNA transcripts (the 'transcriptome'). This technology has revolutionized the study of leukemia, giving insight into genes and pathways involved in disease response and the biology involved in specific translocations.

Acute Disease↗

PubMed2006

Microarray comparative genomic hybridization reveals genome-wide patterns of DNA gains and losses in post-Chernobyl thyroid cancer.

Genetic gains and losses resulting from DNA strand breakage by ionizing radiation have been demonstrated in vitro and suspected in radiation-associated thyroid cancer. We hypothesized that copy number deviations might be more prevalent, and/or occur in genomic patterns, in tumors associated with presumptive DNA strand breakage from radiation exposure than in their spontaneous counterparts. We used cDNA microarray-based comparative genome hybridization to obtain genome-wide, high-resolution copy number profiles at 14,573 genomic loci in 23 post-Chernobyl and 20 spontaneous thyroid cancers. The prevalence of DNA gains in tumors from cases in exposed individuals was two- to fourfold higher than for cases in unexposed individuals and up to 10-fold higher for the subset of recurrent gains. DNA losses for all cases were low and more prevalent in spontaneous cases. We identified unique patterns of copy variation (mostly gains) that depended on a history of radiation exposure. Exposed cases, especially the young, harbored more recurrent gains that covered more of the genome. The largest regions, spanning 1.2 to 4.9 Mbp, were located at 1p36.32-.33, 2p23.2-.3, 3p21.1-.31, 6p22.1-.2, 7q36.1, 8q24.3, 9q34.11, 9q34.3, 11p15.5, 11q13.2-12.3, 14q32.33, 16p13.3, 16p11.2, 16q21-q12.2, 17q25.1, 19p13.31-qter, 22q11.21 and 22q13.2. Copy number changes, particularly gains, in post-Chernobyl thyroid cancer are influenced by radiation exposure and age at exposure, in addition to the neoplastic process.

Adolescent↗

PubMed2005

Genetic variation in bactericidal/permeability-increasing protein influences the risk of developing rapid airflow decline after hematopoietic cell transplantation.

Innate immunity is involved in the biology of graft versus host disease and common airway diseases. We screened 15 genes in this pathway using a linkage disequilibrium-based approach to identify potential candidate genes that may be involved in the development of airflow obstruction after hematopoietic cell transplantation. Sixty-nine single-nucleotide polymorphisms were selected for assessment in a discovery cohort (n = 363). Significant associations were validated in a validation cohort (n = 209). Expression of the candidate gene was demonstrated by detecting gene transcript and protein in malignant and normal small airway epithelial cells. In the discovery cohort, 133 patients developed significant airflow decline. Four patient and donor bactericidal/permeability-increasing (BPI) haplotypes were associated with a 2-fold to 3-fold increased risk of developing significant airflow decline (P values, .004-.038). This association was confirmed in the validation cohort, which had 66 patients with significant airflow decline, with 9 significant haplotypes (P values, .013-.043). BPI gene transcript and protein were detected in airway epithelial cells. These results suggest mutations in the BPI gene significantly influence the risk of developing rapid airflow decline after hematopoietic cell transplantation and may represent a novel therapeutic target for this form of airway disease.

Adult↗

PubMed2005

Analysis of cellular responses to aflatoxin B(1) in yeast expressing human cytochrome P450 1A2 using cDNA microarrays.

Aflatoxin B1 (AFB(1)) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB(1) is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N(7)-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB(1), a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB(1) that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB(1) treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB(1)-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by cell cycle arrest, and suggested that there are additional signaling pathways that directly repress these genes in cells under genotoxic stress.

Aflatoxin B1↗

PubMed2005

A class of models for analyzing GeneChip gene expression analysis array data.

BACKGROUND: Various analytical methods exist that first quantify gene expression and then analyze differentially expressed genes from Affymetrix GeneChip gene expression analysis array data. These methods differ in the choice of probe measure (quantification of probe hybridization), summarizing multiple probe intensities into a gene expression value, and analysis of differential gene expression. Research papers that describe these methods focus on performance, and how their approaches differ from others. To better understand the common features and differences between various methods, and to evaluate their impact on the results of gene expression analysis, we describe a class of models, referred to as generalized probe models (GPMs), which encompass various currently available methods. RESULTS: Using an empirical dataset, we compared different formulations of GPMs, and GPMs with three other commonly used methods, i.e. MAS 5.0, dChip, and RMA. The comparison shows that, on a genome-wide scale , different methods yield similar results if the same probe measures are chosen. CONCLUSION: In this paper we present a general framework, i.e. GPMs, which encompasses various methods. GPMs permit the use of a wide range of probe measures and facilitate appropriate comparison between commonly used methods. We demonstrate that the dissimilar results stem primarily from different choice of probe measures, rather than other factors.

Algorithms↗

PubMed2005

Toll-like receptor 4 polymorphisms are associated with resistance to Legionnaires' disease.

The immunogenetic factors that influence susceptibility to pneumonia are poorly understood. Recent studies suggest an association of toll-like receptor 4 (TLR4) polymorphisms with increased susceptibility to some infections. Here, we examined whether polymorphisms in TLR4 influence susceptibility to Legionnaires' disease (LD) by using a case-control study to compare the allele frequencies of two SNPs (A896G and C1196T). Cases (n = 108) were obtained from a LD outbreak in The Netherlands in 1999. Controls were exposed at the same outbreak, did not develop pneumonia, and were either unmatched (n = 421) or matched (n = 89) to patients for age, sex, and geographic residence. Allele 896G was associated with LD susceptibility with a frequency of 6.5% in the combined control group (matched and unmatched) vs. 2.5% in patients [odds ratio (OR) of 0.36, 95% confidence interval (C.I.) 0.14-0.91, P = 0.025]. In the matched control group comparison, allele 896G also showed a protective association with an OR of 0.27 (95% C.I. 0.09-0.75, P = 0.008). An analysis of genotype frequencies (896 AA vs. AG and GG) demonstrated similar protective associations (patient vs. combined control group comparison, OR = 0.35, 95% C.I. 0.14-0.89, P = 0.02; matched control group comparison, OR = 0.25, 95% C.I. 0.09-0.71, P = 0.006). Allele 1196T cosegregated with allele 896G and, thus, had identical associations. Although previous studies suggest that these TLR4 SNPs are associated with an increased risk of infection, this study demonstrates an association with resistance. This protective association illustrates that an innate immune receptor can mediate either beneficial or deleterious inflammatory responses and that these outcomes vary with different pathogens.

Alleles↗

PubMed2005

Phenotypic anchoring of global gene expression profiles induced by N-hydroxy-4-acetylaminobiphenyl and benzo[a]pyrene diol epoxide reveals correlations between expression profiles and mechanism of toxicity.

The goal of this study was to compare changes in gene expression induced by exposure to different carcinogens and to anchor these changes to the induced levels of toxicity and mutagenesis. The human TK6 lymphoblastoid cell line was used as an in vitro model system, and reactive metabolites of two human carcinogens, benzo[a]pyrene and 4-aminobiphenyl, were used as model compounds. We first determined the toxicity of the model compounds N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and benzo[a]pyrene diol epoxide (BPDE) in TK6 cells. BPDE was about 1000-fold more toxic and mutagenic than N-OH-AABP in TK6 cells on a molar basis. We next treated cells with three doses of each compound that resulted in low, medium, and high toxicities (5, 15, and 40%) and harvested cells at different times after exposure. Using comparable levels of toxicity as the phenotypic anchor, we compared the patterns of gene expression induced by each reactive metabolite using printed cDNA microarrays comprising approximately 18,000 human gene/EST sequences. The microarray data from the N-OH-AABP and BPDE treatment groups were compared using self-organizing map clustering algorithms, as well as a statistical regression modeling approach. While subsets of genes indicative of a generalized stress response [Hsp 40 homologue (DNAJ), Hsp70, Hsp105, and Hsp 125] were detected after exposure to both compounds at all concentrations, there were also many differentially regulated genes, including phase I xenobiotic metabolism [e.g., glutathione transferase omega (GSTTLp28) and antioxidant enzymes (Apxl)]. Other differentially regulated genes included those encoding proteins involved in all major DNA repair pathways, including excision repair (e.g., ERCC5), mismatch repair (e.g., MLH3), damage specific DNA binding protein (e.g., DDB2), and cisplatin resistance-associated overexpressed protein (LUC7A, CRA). Differences in the transcriptional response of TK6 cells to N-OH-AABP or BPDE exposure may explain the dramatic differences in the toxicity and mutagenicity of these human carcinogens.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2005

Evaluation of nine strategies for analyzing a cDNA toxicology microarray data set.

Microarray technology with two-color-based cDNA is commonly used for drug development, as well as for a much broader range of biomedical research. Among all the applications, two-group design is probably most commonly used for comparing, e.g., normal and abnormal tissue samples, tissues treated and untreated, or individuals responded and not responded to a drug. Despite the apparent simplicity, there are numerous methods for analyzing such data in a statistically rigorous manner. Here, we discuss nine different analytical strategies, each of which is derived under a set of "reasonable" assumptions. Some of them resemble methods developed for different contexts. In the absence of the truth, investigators should consider underlying assumptions before taking one or more of these strategies for analyzing data from a particular experiment. The issue here is what are the similarities and differences between these analytical strategies. We present these strategies in the context of an actual microarray experiment performed at the U.S. Food and Drug Administration.

Algorithms↗

PubMed2004

Gene expression analyzed by high-resolution state array analysis and quantitative proteomics: response of yeast to mating pheromone.

The transcriptome provides the database from which a cell assembles its collection of proteins. Translation of individual mRNA species into their encoded proteins is regulated, producing discrepancies between mRNA and protein levels. Using a new modeling approach to data analysis, a striking diversity is revealed in association of the transcriptome with the translational machinery. Each mRNA has its own pattern of ribosome loading, a circumstance that provides an extraordinary dynamic range of regulation, above and beyond actual transcript levels. Using this approach together with quantitative proteomics, we explored the immediate changes in gene expression in response to activation of a mitogen-activated protein kinase pathway in yeast by mating pheromone. Interestingly, in 26% of those transcripts where the predicted protein synthesis rate changed by at least 3-fold, more than half of these changes resulted from altered translational efficiencies. These observations underscore that analysis of transcript level, albeit extremely important, is insufficient by itself to describe completely the phenotypes of cells under different conditions.

Computational Biology↗

PubMed2004

Single-stranded linear amplification protocol results in reproducible and reliable microarray data from nanogram amounts of starting RNA.

The range of scientific questions utilizing DNA microarray techniques is limited by the fact that these methods require 5-40 microg of high-quality total RNA. Thus, methods that reliably amplify the starting RNA amount could expand the applicability of DNA microarray technology. We developed a single-stranded linear amplification protocol (SLAP) that combines the reproducibility of in vitro transcription and the amplification robustness of polymerase chain reactions. We compared SLAP to the NIH-IVT amplification protocol. SLAP displayed excellent conservation of the 5'/3' signal and demonstrated the most robust amplification, producing the recommended amounts of biotin-labeled RNA with as little as 0.002 microg of starting RNA. Both SLAP and NIH-IVT methods demonstrated good reproducibility, but SLAP maintained the highest level of reliability with RNA starting amounts of <0.05 microg. These results suggest that SLAP is an excellent alternative to IVT-based amplification protocols when RNA is limited by small sample size.

Biotin↗