Good Quality RNA Research Articles

Optimization of total RNA isolation from Cochlodinium polykrikoides

Dinoflagellates are eukaryotic microorganisms that represent a group of single-celled algae composed of a highly diversified phylum that is well known for displaying an amazing range of ecological adaptation. Cochlodinium polykrikoides is an unarmored dinoflagellate species belonging to the Family Gymnodiniaceae. It is one of the causative agents for harmful algal blooms occurring in the west coast of Sabah, which result in massive economic damages to the aquaculture and mariculture industries. The limited availability of information pertaining to the genetics of C. polykrikoides raises the need for an in-depth study into the expression mechanisms and regulation of the genes of this microorganism. However, isolation of good quality and intact RNA samples from C. polykrikoides seems to be quite a challenge for molecular biologists. Rapid degradation of freshly isolated RNA samples is a bottleneck for researchers to conduct studies on the gene expression and regulation patterns, which requires intact and high-quality RNA samples. In this study, an attempt to establish the best protocol for isolation of good quality and intact RNA from C. polykrikoides was carried out by testing four different total RNA isolation protocols. We conclude that the RNA isolation protocol using the TRIzol reagent produced the best results among the methods tried. The information from this study will allow further research pertaining to the molecular aspects of C. polykrikoides to be conducted with much ease in the future.

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.

Simple technique for RNA purification from mouse inner ear hair cells

Obtaining a good quality of RNA from small population of cells remain an issue. Isolation for a special anatomic location such as inner ear placed in the temporal bone become a challenge, especially in terms of time needed for isolation of living tissue from the bone, which is a key factor to preserve the RNA. Due to limited accessibility to the technologies such as laser dissection, we present a simplified procedure for isolation of good quality of RNA from the inner ear for further studies.

Comparison of different bead-beating RNA extraction strategies: An optimized method for filamentous fungi

Molecular studies, especially in relation to the activity of secondary metabolite gene clusters, require the ability to extract good quality RNA from fungal biomass. This is often hindered by the cell wall structure and endogenous RNase activity in filamentous fungi. There is thus a need for rapid methods for the extraction of good quality RNA for use in microarrays and for quantitative PCR assays. The objective of this study was to examine the use of different systems for the high throughput method to extract intact RNA from filamentous fungi. Two bead beating systems with different motion patterns and speed capacities were tested in the development of the extraction protocol. They were evaluated based on the total RNA yield and overall RNA quality. The high speed bead beating with glass beads associated with an automated purification method gave more than three times higher total RNA yields with less than a quarter of the amount of mycelium required. Furthermore the integrity and overall quality was conserved, with RNA Quality Indicator (RQI) numbers consistently >7.5. This method also reduced cross contamination risks and kept RNA handling to a minimum while still being capable of multiple sample processing, reducing the time required to obtain RNA from filamentous fungi.

Isolation of High Quality RNA from Phyllanthus emblica and Its Evaluation by Downstream Applications

Next generation sequencing is a high-throughput technique widely used for transcriptome profiling. Isolation of high quality RNA is a prerequisite for such large scale transcriptome analysis. Phyllanthus emblica is an important medicinal plant having high amount of metabolites like vitamin C, flavonoids, polyphenolic compounds, tannins, which are responsible for its wondered medicinal properties. High concentration of secondary metabolites like polysaccharides and polyphenols proved to be an obstacle in isolating RNA of good quality. Any compromise with quality of RNA affects the downstream applications and requires extra cleaning steps that further reduce RNA quantity. We have developed a protocol for isolation of high quality RNA from P. embilca. RNA was successfully assessed for downstream applications like reverse transcription polymerase chain reaction, rapid amplification of cDNA ends, mRNA library preparation, and sequencing using HiSeq(™) 2000 sequencing technology. The protocol is simple and can be completed in 4-5 h.

Needle core biopsies provide ample material for genomic and proteomic studies of kidney cancer: Observations on DNA, RNA, protein extractions and VHL mutation detection

The use of needle biopsies in basic research is increasing, and our study provides a comprehensive analysis of their adequacy in genomic and proteomic studies of kidney cancer. Frozen clear cell renal cell carcinoma (ccRCC) needle core biopsies and sections from core biopsies embedded in optimal cutting temperature (OCT) compound were used to extract DNA, RNA and protein. Their integrity was determined using genomic and proteomic analyses. VHL mutation testing was performed on ccRCC biopsies and corresponding tumors using bulk and laser capture microdissection (LCM) extractions for comparison. Adequate amounts of good quality DNA (5.8–13.3 μg/whole core, 0.6–2.7 μg/20 sections), RNA (2.9–11.9 μg/whole core, 0.5–1.3 μg/20 sections) and protein (137.4–444 μg/whole core, 39.9–74.1 μg/20 sections) were obtained from whole core and frozen sections of ccRCC needle biopsies, respectively. We observed VHL sequence mutations in 75% of ccRCC tumors and, in most cases, the same mutations were detected in both tumors and corresponding biopsies. Mutations observed by bulk extractions from tumors and biopsies were also detected by LCM without significant differences between both methodologies. ccRCC needle biopsies provide ample material for genomic and proteomic studies of kidney cancer. They are good representatives of their corresponding tumors for VHL mutation detection using both bulk and LCM extractions. LCM does not increase sensitivity of VHL mutation detection.

Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.

The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.

Abstract 4848: Genetic profile of invasive breast cancer vasculature using laser capture microdissection (LCM) to capture vessels

Abstract Tumor angiogenesis is vital for tumor growth and progression. Defining genes that are involved in tumor driven angiogenesis may yield therapeutically important targets for cancer therapy. We have developed and optimized a rapid 25 minute immunohistochemical staining method using CD31 antibody (Pecam1) specific to endothelial cells in vessels on sections from frozen human breast cancer (n=3) and normal breast (n=3), followed by laser capture microdisection (LCM) of vessels. Good quality RNA was extracted amplified, and fluorescently labeled cDNA was synthesized and hybridized onto Affymetrix U-133 Plus 2.0 array. Using this methodology, we were able to elucidate the genetic profile of tumor associated vessels from invasive breast cancer and compare it with vessels in normal breast tissue. This technique has the advantage over other methods in analyzing genetic profile of tissue as it analyses the genetic profile of isolated cell population and eliminates genes expressed by surrounding tissue, allowing for the detection of low expression of genes that were not detected in whole tumor arrays. Using false discovery rate statistical analysis (q<0.05) of microarray data, we identified 64 upregulated and 6 downregulated genes in breast cancer associated vessels. With Ingenuity Pathway analysis the top molecular and cellular functions include 11 genes involved in Cellular Assembly and Organization, 9 in Cellular Function and Maintenance and 8 in Protein Synthesis. Canonical pathway analysis revealed 3 genes involved in Ephrin Receptor signaling, 3 in Integrin signaling, 2 in VEGF signaling and 2 in TGF-β signaling. Differentiated genes were compared to Expression Sequence Tags from endothelial and non-endothelial libraries; three genes were specific to endothelial cells only (q<0.05). Using Immunohistochemistry we have validated expression of two genes in breast vessels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4848. doi:10.1158/1538-7445.AM2011-4848

Abstract 4150: CK-19, MAGE-A3, HER-2 and TWIST-1 expression by RT-qPCR in CTCs of primary breast cancer patients after positive immune-magnetic selection using anti-EpCAM and anti-MUC1 magnetic beads

Abstract Background: Circulating tumor cells (CTCs) are suggested as potential surrogate markers for minimal residual disease, the precursor of metastatic disease. Furthermore, to be able to disseminate and metastasize, CTCs must be able to perform epithelial-mesenchymal transition (EMT). The aim of our study was to evaluate the sensitivity of RT-qPCR for the detection and characterization of CTCs analyzing the expression of CK-19, MAGE-A3, HER-2 and TWIST-1 as a transcription factor for EMT in blood of early breast cancer patients after positive immunomagnetic selection using a combination of anti-EpCAM and anti-MUC1 magnetic beads. Patients and Methods: Two × 5 ml blood samples were collected before surgery and CTCs were isolated by positive immunomagnetic selection using anti-EpCAM and anti-MUC1 coated magnetic beads (AdnaTest Breast Cancer Select, AdnaGen AG, Langenhagen, Germany). CK-19, MAGE-A3, HER-2 and PBGD transcripts were simultaneously quantified by multiplex RT-qPCR while TWIST-1 transcript by single RT-qPCR in the LightCycler 2.0 (Roche Diagnostics). All primers and probes were designed to match the assay conditions, such as amplicon sizes, and melting temperatures. Before proceeding to patients’ samples, the RT-qPCR assay was evaluated in respect to sensitivity, specificity, and reproducibilty. To ensure that amplifiable material was present in all specimens and to avoid false-negative results, real-time amplification of the reference gene porphobilinogen deaminase (PBGD) was performed for all samples. A total number of 436 samples were first evaluated for RNA quality by PBGD expression and finally 293 samples that were of good RNA quality were further processed. Results: CK-19 was detected in 117/293 (39.9%), HER-2 in 35/293 (12.0%), MAGE-A3 in 25/293 (8.5%), and TWIST-1 in 16/293 (5.5 %) of all samples, respectively. One gene was detected in 135/293 (46.1%) of the patients, two genes in 23/293 (7.8%) of the patients, three genes in 3/293 (1.0%) patients and four genes in 0/293 (0 %) of the patients, respectively. In total, 161/293 (54.9%) patients were found positive for at least one gene expression, while in 132/293 (45.05%) patients we didn't detect CTCs at all. Discussion: Our study has revealed a high sensitivity when using a combination of immunomagnetic separation and RT-qPCR, as well as a remarkable heterogeneity of gene expression for these genes between early breast cancer patients. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4150. doi:10.1158/1538-7445.AM2011-4150

Next‐generation sequencing of transcriptomes: a guide to RNA isolation in nonmodel animals

Next Generation Sequencing technologies (NGS) are rapidly invading many evolutionary and ecological fields, such as phylogenomics, molecular evolution, population genomics and molecular ecology. Among the potential targets of NGS is transcriptome sequencing, a fast and relatively cheap way to generate massive amounts of coding sequence data, offering promising perspectives for the analysis of molecular diversity in the wild. A number of molecular ecology research groups therefore may switch from DNA-based to RNA-based typing in the near future. Sample preparation from natural populations, however, requires specific care and protocols when RNA is the target. Furthermore, NGS sequencing of transcriptome requires high amount of good-quality RNA. Here we present the results of RNA extraction experiments from various samples of 39 animal species caught in the wild. We compared tissue preparation and storage conditions, evaluated and improved standard RNA extraction protocols, and achieved RNA yield and quality suitable for NGS in all cases. We derive general guidelines for the production of ready-to-sequence RNA in nonmodel animals sampled in the field.

Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

BackgroundTechnical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.

Reduced Dicer expression in the cord blood of infants admitted with severe respiratory syncytial virus disease

BackgroundRespiratory syncytial virus (RSV) is one of the most important causes of pediatric hospital admissions in the developed world. The ribonuclease Dicer is an important regulator of gene expression and cellular function via RNA interference, and may also have anti-viral functions. A previous microarray analysis of the cord blood of 5 patients with RSV disease suggested downregulation of Dicer. In order to further investigate whether reduced Dicer expression can predispose newborns to RSV disease, we have analyzed the gene expression of Dicer in the cord blood of 37 infants with confirmed RSV disease.MethodsThe cord blood of 2108 newborns was collected. 51 had a positive nasopharyngeal aspirate for RSV <1 year, and were grouped according to disease severity. 37 had sufficient cord blood RNA of good quality. Dicer gene expression was assessed by qPCR analysis of cord blood using a TaqMan low-density array and compared to control infants who did not present with RSV disease using the Mann-Whitney test.ResultsThere was significant downregulation of Dicer in the severe disease group: relative quantity 0.69 (95% CI: 0.56 - 0.87), p = 0.002. There was no significant downregulation in the mild disease group.ConclusionsWe demonstrate reduced Dicer expression in the cord blood of infants with severe RSV disease, prior to RSV exposure. We theorize that this may predispose to RSV disease by disruption of leukocyte gene regulation or direct anti-viral RNA interference mechanisms.

Isolation of RNA from Field-Grown Jute (Corchorus capsularis) Plant in Different Developmental Stages for Effective Downstream Molecular Analysis

Jute (Corchorus capsularis), as a natural fibre producing plant species, ranks next to cotton only. Today, biotechnological approach has been considered as most accepted means for any genetic improvement of plant species. However, genetic control of the fibre development in jute has not yet been explored sufficiently for desired genetic improvement. One of the major impediments in exploring the genetic architecture in this crop at molecular level is the availability of good quality RNA from field-grown plant tissues mostly due to the presence of high amount of mucilage and phenolics. Development of a suitable RNA isolation method is becoming essential for deciphering developmental stage-specific gene expression pattern related to fibre formation in this crop species. A combination of modified hot borate buffer followed by isopycnic centrifugation (termed as HBIC) was adopted and found to be the best isolation method yielding sufficient quantity (~350-500 μg/gm fresh tissue) and good quality (A(260/280) ratio 1.88 to 1.91) RNA depending on the developmental stage of stem tissue from field-grown jute plant. The poly A(+) RNA purified from total RNA isolated by the present method was found amenable to efficient RT-PCR and cDNA library construction. The present development of RNA isolation was found to be appropriate for gene expression analysis related to fibre formation in this economically important jute plant in near future.

Improvements in the re-flight of spaceflight experiments on plant tropisms

In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1- g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.

Cyclooxygenase-2 network as predictive molecular marker for clinical pregnancy in in vitro fertilization

The gene expression of human endometrium on the day of oocyte retrieval of pregnant and nonpregnant patients in a stimulated IVF cycle was compared in two independent groups with different GnRH-antagonist ovarian stimulation protocols. The present data suggest that increased gene expression of cyclooxygenase-2, together with the expression of other molecules in the cyclooxygenase-2 network, on the day of oocyte retrieval in GnRH-antagonist cycles coincides with a lower probability of achieving a clinical pregnancy in this cycle.

Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis

The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.

Abstract 4672: Tumor dihydropyrimide dehydrogenase (DPYD) expression is associated with survival in patients with metastatic colorectal cancer treated with gefitinib, 5-fluorouracil, leucovorin, and oxaliplatin (IFOX)

Abstract New drugs that target the EGFR signaling pathways suggest that EGFR inhibition exerts a chemosensitizing effect in colorectal cancer. We have previously reported that the combination of gefitinib, leucovorin, 5-flourouracil, and oxaliplatin (IFOX) resulted in higher response rates than those reported with FOLFOX-4 alone in patients with metastatic colorectal cancer. The aim of the present study was to examine genomic factors that might be associated with response or survival in the IFOX regimen. Patients and Methods: Seventy-two patients had stage IV colorectal adenocarcinoma and were either untreated or previously treated prior to receiving the IFOX regimen. Formalin-fixed, paraffin-embedded tissues (FFPE; n=63) were obtained, and 4-um sections were cut, placed on slides, and deparaffinized in xylene, followed by laser capture microdissection (LCM; n=42) to enrich for tumor cells. Using real-time quantitative RT-PCR, thirty-five samples had good-quality actin RNA readings following LCM, and the expression levels of dihydropyrimidine dehydrogenase (DPYD), epidermal growth factor receptor (EGFR), excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1), thymidylate synthase (TYMS), and thymidine phosphorylase (TYMP) were separately quantified. To detect mutations in KRAS, DNA was extracted from fifty-eight patient samples and analyzed using the KRAS Mutector II kit (TrimGen, Sparks, MD). Results: For the 33 patients available for RT-PCR analysis, expression levels of DPYD, EGFR, ERCC1, TYMP, or TYMS did not correlate significantly with response. However, in terms of survival, the overall survival rate was significantly worse in patients with a high DPYD expression level compared to patients with a low DPYD expression level (p&amp;lt;0.05), and in a multivariate analysis, patients with both a high DPYD expression and a low EGFR expression had the worst survival compared to all other patients with differing expression levels of these two genes (p&amp;lt;0.01). KRAS mutations were identified in 24 out of 58 patients (41%), but KRAS mutational status did not correlate with either response or survival. Conclusion: Dihydropyrimidine dehydrogenase mRNA expression was a significant independent prognostic factor for overall survival in colorectal cancer patients treated with FOLFOX-4 along with EGFR inhibition by gefitinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4672.

Abstract A7: Combined copy number and expression analysis of formalin fixed-paraffin embedded (FFPE) tumours

Abstract Introduction: FFPE tissues are a valuable source of tumour material for translational research that has thus far been under-used. But progress in technology has allowed the analysis of genome wide copy number and expression changes using small amounts of degraded DNA/RNA extracted from FFPE tissues. We used two platforms: molecular inversion probes assay (MIP, Affymetrix) and c-DNA mediated Annealing Selection extension and Ligation assay (DASL, Illumina™) for analysis of RNA/DNA extracted from routine core biopsies from a clinical trial to identify molecular markers of response or resistance to chemotherapy. Method: FFPE diagnostic core biopsies of 63 patients treated with neoadjuvant adriamycin/cyclophosphamide followed by taxotere in a clinical study were used. MIP assay with 50K SNP panel was used for copy number analysis (CNA). DASL assay assessed expression of 502 genes using three probes per gene. Results: RNA and DNA of good quality were extracted from 52/63 (82%) and 46/63 (73%) core biopsy samples, respectively. Gene expression profiling was carried out using the DASL assay and there were good correlations with IHC for ER, PR and HER-2 with area under the curve of 0.95, 0.90 and 0.96 respectively, following ROC analysis for the best transcripts. Differential expression analysis between ER+ vs. ER- cancers showed that ESR1, TFF1 and LAF4 are significantly over-expressed in ER + cancers (p&amp;lt;0.001), while analysis of pathological complete responders vs. non responders showed over expression of CXCL9, ARHA, ARHGDIB, BCL3, ERBB2, BMYB and TGFBI among responders (p&amp;lt;0.2). CNA showed well described aberrations in breast cancer such as high probability of gains in 1q, 8q, 11q and 20q and loss in 4q, 12q, 13q, 18q and 22q (probability of alteration ≥0.35). Samples with pathological complete response had high probability of gains in 1q (55%), 8q (40%), 17q (40%) and losses in 8p (40%) and 13q (70%). 5/46 samples were HER-2 3+ with IHC and all showed amplification around the 17q locus. Conclusions: Reliable copy number and expression data can be generated using these assays from FFPE core biopsy samples. Role of LAF4 as an oestrogen regulated gene and roles of CXCL9, ARHA and ARHGDIB as markers of response to chemotherapy should be further investigated. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A7

Gene expression profiling in psoriatic scalp hair follicles: clobetasol propionate shampoo 0.05% normalizes psoriasis disease markers

Clobetasol propionate shampoo is effective and safe in treatment of scalp psoriasis (SP). Gene expression profiling of psoriatic skin biopsies led to the identification of numerous disease-related genes. However, it remained unknown whether the gene expression profile of hair follicles of SP patients was also affected. To determine whether psoriasis-related genes are differentially regulated in the hair follicles of SP patients and whether the modulation of these genes can be correlated with clinical severity scores. A single arm, open study was conducted in three centres. SP patients received daily treatment with clobetasol propionate shampoo. At Baseline, Weeks 2 and 4, investigators assessed clinical severity parameters and collected scalp hair follicles in anagen phase. Total RNA extracted from hair follicles was used to determine the expression level of 44 genes, which were reported previously to be upregulated in the skin of psoriasis patients. RNA of good quality and sufficient quantity was obtained from hair follicles of psoriasis patients and healthy volunteers (HV). The expression level of 10 inflammation-related genes was significantly increased in psoriatic hair follicles. The patient's exploratory transcriptomic score, defined as the mean fold modulation of these 10 genes compared with HV, correlated with clinical severity scores. Clobetasol propionate shampoo was effective in decreasing both the exploratory transcriptomics and the clinical severity scores. Hair follicles of SP patients are affected by the inflammatory process. The change in the expression level of inflammation-related genes correlates with the severity of the disease.