Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study

BACKGROUND: Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip(® )technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology. RESULTS: The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results. CONCLUSIONS: This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance

A Spray-on, Nanocomposite-Based Sensor Network for <i>in-Situ </i>Active Structural Health Monitoring

A new breed of nanocomposite-based spray-on sensor is developed for in-situ active structural health monitoring (SHM). The novel nanocomposite sensor is rigorously designed with graphene as the nanofiller and polyvinylpyrrolidone (PVP) as the matrix, fabricated using a simple spray deposition process. Electrical analysis, as well as morphological characterization of the spray-on sensor, was conducted to investigate percolation characteristic, in which the optimal threshold (~0.91%) of the graphene/PVP sensor was determined. Owing to the uniform and stable conductive network formed by well-dispersed graphene nanosheets in the PVP matrix, the tailor-made spray-on sensor exhibited excellent piezoresistive performance. By virtue of the tunneling effect of the conductive network, the sensor was proven to be capable of perceiving signals of guided ultrasonic waves (GUWs) with ultrahigh frequency up to 500 kHz. Lightweight and flexible, the spray-on nanocomposite sensor demonstrated superior sensitivity, high fidelity, and high signal-to-noise ratio under dynamic strain with ultralow magnitude (of the order of micro-strain) that is comparable with commercial lead zirconate titanate (PZT) wafers. The sensors were further networked to perform damage characterization, and the results indicate significant application potential of the spray-on nanocomposite-based sensor for in-situ active GUW-based SHM

Comparative evaluation of an extensive histopathologic examination and a real-time reverse-transcription-polymerase chain reaction assay for mammaglobin and cytokeratin 19 on axillary sentinel lymph nodes of breast carcinoma patients

OBJECTIVE: To assess the accuracy of a commercially available real-time reverse-transcription-polymerase chain reaction assay for mammaglobin and cytokeratin 19 mRNAs [GeneSearch Breast Lymph Node (BLN) Assay, Veridex LLC, Warren, NJ] in the detection of axillary sentinel lymph nodes (SLNs) metastases in patients with breast carcinoma. SUMMARY BACKGROUND DATA: Because of the lack of standardized and widely accepted protocols for a truly accurate histopathologic examination of SLN, the relative merits of alternative assays based on the identification of tumor specific mRNA markers deserve further assessment. METHODS: A prospective series of 293 consecutive SLNs from 293 patients was evaluated. The BLN assay results were compared with those of an extensive histopathologic examination of the entire SLNs performed on serial frozen sections cut at 40 to 50 μm intervals. RESULTS: The BLN assay correctly identified 51 of 52 macrometastatic and 5 of 20 micrometastatic SLNs, with a sensitivity of 98.1% to detect metastases larger than 2 mm, 94.7% for metastases larger than 1 mm, and 77.8% for metastases larger than 0.2 mm. The overall concordance with histopathology was 90.8%, with specificity of 95.0%, positive predictive value of 83.6%, and negative predictive value of 92.9%. When the results were evaluated according to the occurrence of additional metastases to non-SLN in patients with histologically positive SLNs, the assay was positive in 33 (91.7%) of the 36 patients with additional metastases and in 22 (66.6%) of the 33 patients without further echelon involvement. CONCLUSIONS: The sensitivity of the reverse-transcription -polymerase chain reaction assay is comparable to that of the histopathologic examination of the entire SLN by serial sectioning at 1.5 to 2 mm. © 2008 Lippincott Williams & Wilkins, Inc