Rounds Of Amplification Research Articles

Identification, Diversity and Evolution of MITEs in the Genomes of Microsporidian Nosema Parasites.

Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous DNA transposons, which are widespread in most eukaryotic genomes. However, genome-wide identification, origin and evolution of MITEs remain largely obscure in microsporidia. In this study, we investigated structural features for de novo identification of MITEs in genomes of silkworm microsporidia Nosema bombycis and Nosema antheraeae, as well as a honeybee microsporidia Nosema ceranae. A total of 1490, 149 and 83 MITE-related sequences from 89, 17 and five families, respectively, were found in the genomes of the above-mentioned species. Species-specific MITEs are predominant in each genome of microsporidian Nosema, with the exception of three MITE families that were shared by N. bombycis and N. antheraeae. One or multiple rounds of amplification occurred for MITEs in N. bombycis after divergence between N. bombycis and the other two species, suggesting that the more abundant families in N. bombycis could be attributed to the recent amplification of new MITEs. Significantly, some MITEs that inserted into the homologous protein-coding region of N. bombycis were recruited as introns, indicating that gene expansion occurred during the evolution of microsporidia. NbS31 and NbS24 had polymorphisms in different geographical strains of N. bombycis, indicating that they could still be active. In addition, several small RNAs in the MITEs in N. bombycis are mainly produced from both ends of the MITEs sequence.

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast.

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Antisense RNA amplification for target assessment of total mRNA from a single cell.

This protocol describes how to amplify mRNA isolated from a single cell and then analyze its gene expression profile using polymerase chain reaction (PCR). Single-cell analysis is advantageous over studies of cell populations because it allows identification of a range of normal physiological states expressed by different cells of the same cell type without the confounding effects of averaging that result from measuring physiological states of cell populations. This is especially important when addressing questions of physiology in tissues, which comprises many different cell types. However, a single cell does not contain enough mRNA for all of the expressed transcripts to be detected or measured by any current molecular biology techniques. The antisense RNA (aRNA) amplification method was developed to amplify the picogram amounts of mRNA found within a single cell to microgram amounts of aRNA after three rounds of amplification. This aRNA can then easily be analyzed by microarray or next-generation sequencing. These methods allow identification of all expressed mRNA species within a single cell, including previously unknown mRNAs or those mRNAs specifically affected by a certain treatment. mRNA species of interest identified by these techniques can be further analyzed by designing primers targeting these species and performing PCR. cDNA synthesized from RNA at any stage in the aRNA amplification procedure, including material directly from collected unamplified cells, can be analyzed using PCR. Regardless of downstream applications, single-cell aRNA amplification is a powerful tool for studying single-cell physiological dynamics.

Improved detection of malaria cases in island settings of Vanuatu and Kenya by PCR that targets the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene

Detection of sub-microscopic parasitemia is crucial for all malaria elimination programs. PCR-based methods have proven to be sensitive, but two rounds of amplification (nested PCR) are often needed to detect the presence of Plasmodium DNA. To simplify the detection process, we designed a nested PCR method whereby only the primary PCR is required for the detection of the four major human Plasmodium species. Primers designed for the detection of the fifth species, Plasmodium knowlesi, were not included in this study due to the absence of appropriate field samples. Compared to the standard 18S rDNA PCR method, our cytochrome c oxidase III (cox3) method detected 10–50% more cases while maintaining high sensitivities (1.00) for all four Plasmodium species in our samples from Vanuatu (n=77) and Kenya (n=76). Improvement in detection efficiency was more substantial for samples with sub-microscopic parasitemia (54%) than those with observable parasitemia (10–16%). Our method will contribute to improved malaria surveillance in low endemicity settings.

Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

BackgroundEstablishing highly productive clonal cell lines with constant productivity over 2–3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure.ResultsWe have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative.ConclusionsThe p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

Construction of recombinant adenovirus vector co-expressing VEGF165 and SDF-1 genes and its expression in ischemic cerebral tissue of rats

To construct a recombinant adenoviral vector carrying and co-expressing vascular endothelial growth factor 165 (VEGF165) and stromal cell derived factor 1 (SDF-1) and explore its co-expression in ischemic brain tissue in rats. The VEGF165 and SDF-1 genes were directionally connected with internal ribosome entry site (IRES). And the double gene co-expression recombinant shuttle plasmid pDC316-VEGF165-IRES-SDF-1 was built with homologous recombination. The resultant plasmid pDC316-VEGF165-IRES-SDF-1 and backbone plasmid pBHGlox_E1, 3Cre were transfected into HEK293 cells by liposome and the recombinant adenoviral particles capable of infection were acquired. With the rounds of amplification, the purified adenoviral vector Ad5-VEGF165-IRES-SDF-1 was obtained with a titer of up to 1×10(10) IU/ml. The rat model of middle cerebral artery occlusion (MCAO) was established by intra-luminal suturing. And the viral vectors were transfused into the lateral ventricle by a stereotactic microinjection. The expressions of VEGF165 and SDF-1 in ischemic brain tissue were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The results of PCR, double enzyme digestion and gene sequencing showed that both the recombinant plasmid and the constructed adenoviral vector were expressed. And the adenoviral vector Ad5-VEGF165-IRES-SDF-1 could mediate a co-expression of VEGF165 and SDF-1 in ischemic cerebral tissue. The recombinant adenoviral vector carrying VEGF165 and SDF-1 are successfully constructed. And Ad5-VEGF165-IRES-SDF-1 may mediate a co-expression of VEGF165 and SDF-1 in ischemic cerebral tissue of rats.

First Detection of Spring Viremia Of Carp Virus in Cyprinus Carpio, In Lake Shkodra/Scadar Albania

Spring viremia of carp is a highly contagious viral disease caused by Rhabdovirus carpio which is a single strand RNA virus. A combination of one step RT-PCR (reverse transcription) and Semi-nested PCR was used to detect spring viremia of carp virus in infected cell cultures (EPC) and fish tissues. All the fish samples were collected from Lake Shkodra and from artificial ponds in different region of Albania. Total RNA was extracted from 500 μl of fish tissue extract or from 100 μl of supernatant from infected EPC cell cultures. One step RT-PCR is performed using a modification of the methods of Stone et al.(1) in a 15 μl reaction mix volume. We used SVCV F1 (primer forward) 5’- TCT-TGG-AGC-CAA-ATA-GCT-CAR*-R*TC-3’ and SVCV R2 ( primer reverse) 5’-AGA-TGG-TAT-GGACCC- CAA-TAC-ATH*-CAN*-CAY*-3 primers for the amplification of 714 bp fragment of SVCV cDNA (2). The reaction mix was subjected to thermal cycles (2). Amplified cDNA was analyzed in agarose gel electrophoresis. For the second round of amplification we used a semi-nested PCR with SVCV F1 5’-TCT-TGG-AGC-CAA-ATA-GCT-CAR*- R*TC-3’ and SVCV R4 5’-CTG-GGG-TTT-CCN*-CCT-CAA-AGY*-TGY*-3* primers (1). Amplified fragment cDNA (606 bp) is analyzed by agarose gel electrophoreses (2).The results of gel electrophoreses was positive for three fish samples (Cyprinus carpio) collected from Lake Shkodra. This is the first report of the detection of spring viremia of carp virus in Albania in a natural habitat like Lake Shkodra.

Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group

PurposeWe have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification.ResultsThe overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/106 PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /106 PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification.ConclusionsOur results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome

Digital polymerase chain reaction (PCR) is an emerging absolute quantification method based on the limiting dilution principle and end-point PCR. This methodology provides high flexibility in assay design without influencing quantitative accuracy. This article describes an assay to quantify HIV DNA that targets a highly conserved region of the HIV-1 genome that hampers optimal probe design. To maintain high specificity and allow probe binding and hydrolysis of a probe with low melting temperature, a two-stage touchdown PCR was designed with a first round of amplification at high temperature and a subsequent round at low temperature to allow accumulation of fluorescence.

Detection of Hepatitis B Virus Pre-core Mutant by Allele Specific Polymerase Chain Reaction

Introduction : Mutation in pre-core region is characterized by negative HBeAg and positive anti-HBe despite active replications of the virus. The mutation has diagnostic and prognostic implications. Therefore, detection of pre-core mutant is important. Standard diagnosis approach for detecting pre-core mutant is through DNA sequencing of hepatitis B virus (HBV) pre-core region. Unfortunately, DNA sequencing is not available in most centers. Hence, a simpler diagnostic approach is necessary. Method : An observational-analytic design study was performed. Detection of pre-core mutant was conducted in individuals with positive HBsAg and HBV DNA that had various patterns of HBeAg and anti HBe. HBsAg, HBeAg and anti-HBe was detected using immunochromatography technique. The HBV DNA was evaluated by using qualitative polymerase chain reaction (PCR) testing. PCR was done by three rounds of amplification with primers derived from wild type pre-core and mutant pre-core. Results : Of 25 sera with HBeAg negative, anti-HBe positive and HBV DNA positive, allele specific (AS) PCR pre-core mutant was detected in 20 (80%) sera. Two sera with HBeAg negative, anti HBe negative and HBV DNA positive were negative for pre-core mutant. Of 8 sera with HBeAg positive, anti HBe negative and HBV DNA positive, pre-core mutant was detected in 2 (25%) sera. Conclusion : Most of individuals with HBV DNA positive, HBeAg negative and anti-HBe positive have harbored pre-core mutant. The finding indicated that all patients with HBsAg positive, HBV DNA positive and HBeAg negative, but anti-HBe positive should be examined for the presence of pre-core mutant. Pre-core mutant is also found in HBeAg positive individual. Keywords : HBV, pre-core mutant, polymerase chain reaction

Development of a multiplex polymerase chain reaction‐sequence‐specific primer method for NKG2D and NKG2F single‐nucleotide polymorphism typing using isothermal multiple displacement amplification products

Natural killer group 2 member D (NKG2D) on immune effector cells recognizes multiple stress-inducible ligands. NKG2D single-nucleotide polymorphism (SNP) haplotypes were related to the levels of cytotoxic activity of peripheral blood mononuclear cells. Indeed, these polymorphisms were also located in NKG2F. Isothermal multiple displacement amplification (IMDA) is used for whole genome amplification (WGA) that can amplify very small genomic DNA templates into microgram with whole genome coverage. This is particularly useful in the cases of limited amount of valuable DNA samples requiring multi-locus genotyping. In this study, we evaluated the quality and applicability of IMDA to genetic studies in terms of sensitivity, efficiency of IMDA re-amplification and stability of IMDA products. The smallest amount of DNA to be effectively amplified by IMDA was 200 pg yielding final DNA of approximately 16 µg within 1.5 h. IMDA could be re-amplified only once (second round of amplification), and could be kept for 5 months at 4°C and more than a year at -20°C without loosing genome coverage. The amplified products were used successfully to setup a multiplex polymerase chain reaction-sequence-specific primer for SNP typing of the NKG2D/F genes. The NKG2D/F multiplex polymerase chain reaction (PCR) contained six PCR mixtures for detecting 10 selected SNPs, including 8 NKG2D/F SNP haplotypes and 2 additional NKG2D coding SNPs. This typing procedure will be applicable in both clinical and research laboratories. Thus, our data provide useful information and limitations for utilization of genome-wide amplification using IMDA and its application for multiplex NKG2D/F typing.

Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle

Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle

High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing

ObjectiveTo identify disease-causing mutations within coding regions of 11 known NPHP genes (NPHP1-NPHP11) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost.MethodsMutation analysis was carried...

Desarrollo de un método de transcripción reversa seguida de reacción en cadena de la polimerasa para la detección del virus de la fiebre amarilla

Introduction. Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. Objective. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. Materials and methods. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. Results. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. Conclusion. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures. doi: http://dx.doi.org/10.7705/biomedica.v33i0.1452

Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification

Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.

Evaluation of a new single tube RNA amplification method with low 3′/5′ ratios

Expression microarrays have become a common tool in basic and clinical biomedical sciences. However, current technology is limited by the need for relatively large amounts of RNA for labeling and hybridization, or multiple rounds of amplification are required that may reduce the quality of resulting RNA. Methods in use today for amplification of low input RNA often under represent the 5′ end of mRNAs. In this study, we have developed a unique one tube T7 based RNA amplification kit that can reproducibly amplify nanogram amounts of total RNA and generate gene expression profiles that are independent of amount of input RNA, and are comparable with larger amounts of RNA amplified by other accepted methods. The actin 3′/5′ ratio remains low whether amplifying and labeling 20 ng or 500 ng total RNA. The resulting labeled RNA gives high %P calls on Affymetrix arrays, and provides low 3′/5′ ratios even with low input RNA.

Orthogonal Amplification of Nanoparticles for Improved Diagnostic Sensing

There remains an ongoing need for fast, highly sensitive, and quantitative technologies that can detect and profile rare cells in freshly harvested samples. Recent developments in nanomaterial-based detection platforms provide advantages over traditional approaches in terms of signal sensitivity, stability, and the possibility for performing multiplexed measurements. Here, we describe a bioorthogonal, nanoparticle amplification technique capable of rapid augmentation of detection sensitivities by up to 1-2 orders of magnitude over current methods. This improvement in sensitivity was achieved by (i) significantly reducing background noise arising from nonspecific nanoparticle binding, (ii) increasing nanomaterial binding through orthogonal rounds of amplification, and (iii) implementing a cleavage step to improve assay robustness. The developed method allowed sensitive detection and molecular profiling of scant tumor cells directly in unpurified human clinical samples such as ascites. With its high sensitivity and simplified assay steps, this technique will likely have broad utility in nanomaterial-based diagnostics.

Nested RT-PCR for ante mortem diagnosis of rabies from body secretionexcretion of animals suspected for rabies

Aim: The present study deals with molecular technique Nested RT-PCR for detection of rabies viral RNA from biological fluid samples (Saliva, Milk and Urine) collected from animal suspected for rabies and to compare the sensitivity of Nested RT-PCR applied for ante mortem diagnosis of rabies with conventional technique (immunofluorescence) applied on neural tissue. Molecular technique Materials and Methods: Nested RT-PCR was applied on 62 biological fluid specimens collected from rabies suspected animals. First round amplification with nested set of primers (RabN1 and RabN5) yielded 1477 bp product while amplification with second round primers (RabNfor and RabNrev) yielded 762 bp product. Sensitivity of the technique was compared in accordance with WHO recommended gold standard test viz. Immunofluorescence (FAT) applied on brain samples. Results: By Nested RT-PCR, viral RNA could be detected in 9/24 (37.50%) saliva samples, 2/17 (11.76%) milk samples and 6/21 (28.57%) urine samples. Confirmatory diagnosis by Immunofluorescence performed on brain sample revealed 18 true positive cases. Overall, Sensitivity of Nested RT-PCR technique employed on fluid samples was 69.23% when compared with immunofluorescence performed on brain samples. Conclusions: Early reliable ante mortem diagnosis of rabies can be obtained from biological fluid samples of animals suspected to be rabid when tested with Nested RT-PCR technique. Key-words: milk,nested RT-PCR, rabies, saliva, urine

Intravitam Diagnosis of Rabies from Saliva by Nested RT-PCR

In the present study, rabies was confirmed by Nested RT-PCR on saliva of 24 animals suspected to be rabid. Amplification with first round primers (Rab N1 and Rab N5) yielded a 1477 bp product, while primers used for second round amplification (RabNfor and RabNrev) yielded a 762 bp product. By nested RT-PCR, viral RNA could be detected in 9/24 (36.0%) saliva samples. Confirmatory diagnosis by Immunofluorescence performed on brain sample revealed 17 true positive cases. Sensitivity of 68.0% was recorded by application of Nested RT-PCR on saliva samples. It was concluded that Nested RT-PCR applied on saliva of rabies suspected animal can help in intravitam diagnosis of rabies.

P3-06-06: Comparison of Gene Expression Profiles of Lymph Node Positive and Lymph Node Negative ER Positive Breast Tumors in Pre- and Postmenopausal Women.

Abstract Background Breast cancer is the most common female cancer in US and is the second leading cause of cancer related death in women. Metastases are the primary cause of cancer morbidity and mortality. Axillary lymph node (LN) status has long been used as a prognostic factor for breast cancer. The molecular mechanisms that control LN metastasis remains poorly understood. To better understand the various genes and regulatory pathways that drive breast cancer LN metastasis, we compared the gene expression profiles between breast tumors that have metastasized to the LNs and those which have not in pre- and postmenopausal women. Material and Methods: Tumor cells were isolated from the primary tumors (ER+) of postmenopausal node positive (PMNP; N=20), postmenopausal node negative (PMNN; N=19), premenopausal node positive (PRNP; N=18) and premenopausal node negative (PRNN; N=16) women using laser capture microdissection. RNA was isolated using the RNAqueous®-micro kit (Ambion, Austin, TX). Total RNA was converted to Biotin-labelled aRNA using two rounds of amplification with MessageAmp II aRNA amplification kit (Applied Biosystems, Foster City, CA). The aRNA concentration was determined by Nanodrop 1000 and the quality was assessed with a Bioanalyzer. The aRNA was fragmented and hybridized to Human Genome U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA). Microarray raw data were analyzed using a variety of R programming packages for probe density processing, background correction, normalization, quality control/quality assessment, and calculation of gene expression value, etc. To identify differentially expressed genes, Wilcoxon rank sum test with FDR (false discovery rate) control was performed for pair-wise comparison between different groups. Functional analyses were performed on the identified statistically significant differentially expressed genes to search for the functional categories and pathways in which they are involved and further understand their potential roles in breast cancer metastatic process. Results: Multivariate data mining (hierarchical clustering analysis and principal component analysis, etc) revealed that in postmenopausal women, the node positive and node negative women are well separated while this was not the case in premenopausal women. Further analysis of the PMNN and PMNP groups to identify differentially expressed genes (with at least a 1.5 fold difference) at FDR =0.1 showed that 232 genes were upregulated and 470 genes were downregulated in PMNP vs PMNN groups. Gene function analysis revealed that genes down regulated in the PMNP group compared to PMNN are related to extracellular matrix, cell adhesion, EGF-like pathway, cytoskeleton etc, while the over-expressed genes are related to cell cycle and cell division, chromosome condensation, etc. Discussion: The ability to differentiate lymph node positive cases from lymph node negative cases in ER+ breast cancer based on transcriptional profiling may have an impact on the clinical management of ER+ breast cancer cases. Having transcriptional profiles that identify ER+ tumors likely to have poor outcomes would suggest more aggressive treatment for such patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-06-06.