Automated Electrophoresis System Research Articles

Assessment of cfDNA levels in saliva samples of stressed young adults: Preliminary study

Purpose: The aim of this pilot study was to examine the levels of cell-free DNA (cfDNA) in the saliva samples of second-year medical students before and after a stress-inducing event, exams. Materials and Methods: Saliva samples were collected from 20 students, whose stress profiles were assessed using the DASS-21 scale, both before and after the exams. Cell-free DNA were isolated from these samples, and measured using spectrophotometry and automated electrophoresis system. Results: Spectrophotometric analysis revealed a significant decrease in cfDNA levels in the saliva samples collected after the exams (p ≤ 0.05). This difference was confirmed by the automated electrophoresis system, particularly showing a reduction in cfDNA amounts in the 40-200 bp range with the reduction of stress (p ≤ 0.05). Conclusion: Psychosocial stress affects the release of cell-free DNA. This study reports that the amount of cfDNA found in saliva samples significantly changes in relation to stress. This preliminary study suggests that cfDNA in saliva samples could potentially serve as a biological marker for stress in healthy individuals.

Concise Analysis of Single-Stranded DNA of Recombinant Adeno-Associated Virus By Automated Electrophoresis System.

Recombinant adeno-associated virus (rAAV) is a prominent viral vector currently available for human gene therapy. The diameter of the rAAV capsid is ∼25 nm, and a positive or negative single-stranded DNA is packaged within the vector capsid. In this report, we describe a concise method to examine the extracted rAAV genome using an automated electrophoresis system. The rAAV genome, prepared from vector particles through either heat treatment at 95°C for 10 min or the phenol-chloroform extraction method, was analyzed using an automated electrophoresis system under denaturation conditions. The heat treatment protocol demonstrated a comparable yield with the phenol-chloroform extraction protocol, and the quantified amounts of the rAAV genome obtained using the automated electrophoresis system were consistent with those quantitated by quantitative PCR. Additionally, crude rAAV extractions could also be analyzed by the automated electrophoresis system after DNase I treatment. These results indicated that this simple and quick analysis using automated electrophoresis is highly useful for confirming the purity and integrity of the rAAV genome.

Comparative analysis of the DNA isolated from thyme leaves using different methods

Background. The base for a molecular analysis is DNA of high quality. For DNA isolation, different kits or classical methods are used. For mass analysis, isolation with kits is a very expensive process. So, the objective of our investigation was to find a cheap method for high-quality DNA isolation from leaves of various thyme cultivars.Materials and methods. Leaves cut from thyme accessions (Thymus mastichina L. cv. ‘Svetliachok’, T. striatus Vahl. cv. ‘Jubileiniy’, T. vulgaris L. cv. ‘Fantasia’, and T. vulgaris cv. ‘Jalos’.) maintained ex situ in the collection of the Nikita Botanical Gardens were used as the material for the analysis. Light microscopy was used to study leaf anatomy and localize essential oil on leaf cross sections. Essential oil was extracted on Ginsberg devices, and phenolic content was measured with The Folin–Ciocâlteu reagent (FCR). Commercial kits (DiamondDNATM, PureLink® Plant Total DNA Purification Kit) and classical methods (CTAB, CTAB with 2% polyvinylpyrrolidone) were used for DNA isolation. DNA quality was evaluated spectrophotometrically, with electrophoresis (horizontal, automated system Agilent 4200 TapeStation) and PCR.Results. The analysis showed that the leaf blade mesophyll of four thyme cultivars had inclusions with essential oil. The content of essential oil and phenolic compounds was measured biochemically. Since the plants were characterized by the presence of secondary metabolites, DNA was isolated by different methods. Spectrophotometry demonstrated that the classical CTAB method and CTAB with 2% PVP provided the best results. Using an automated electrophoresis system, the presence of high-molecularweight DNA (more than 52000 bp) in significant amounts was detected in the samples isolated with DiamondDNATM kit and CTAB + 2% PVP.Conclusion. Among the tested kits and methods, CTAB + 2% PVP provided thyme DNA suitable for PCR and, presumably, for genome library preparation. The low cost of reagents for this technique makes it applicable for future mass analysis of plant material.

Capillary electrophoresis as alternative method to detect tumor genetic mutations: the model built on the founder BRCA1 c.4964_4982del19 variant.

Despite some populations show a wide spectrum of different BRCA pathogenic variants (PVs), particular ethnic groups carry at high frequency a single or a few recurrent PVs, usually due to a founder effect. The identification of these founder PVs, with simple molecular methods, improves BRCA1/2 testing and cancer risk assessment. In this study, we developed a rapid and reliable PCR method, coupled with capillary electrophoresis (CE) for genotyping the Italian founder BRCA1 c.4964_4982del19 (rs80359876) variant. In addition, we compared the performance of two CE platforms: (Agilent 2100 Bioanalyzer and the Experion Automated Electrophoresis system) to identify this variant. Our findings suggest that CE represents a simple and standardized diagnostic strategy for the unambiguously identification of the BRCA1 c.4964_4982del19 variant, on both germline and somatic DNA samples. The results and performance obtained by two platforms are absolutely superimposable in terms of specificity and sensitivity, as well as for their feasibility, time of analysis and costs.

51 - Implementation of a New Automated Sample Quality Control Tool in a Whole Exome Sequencing Workflow

Objectives: The German Cancer Research Center (DKFZ) is one of the largest biomedical research institutions in Germany. The High Throughput Sequencing Unit of the DKFZ Genomics and Proteomics Core Facility provides sequencing services for multiple applications. This project demonstrates the use of an automated electrophoresis system as a quality control (QC) tool in a whole exome sequencing workflow. Mandatory for the experimental success of whole exome sequencing is the quality of the incoming genomic DNA (gDNA) material and the DNA samples at various stages of the library preparation workflow. Methodology: Exome libraries were prepared according to the Agilent Low Input Sure-SelectXT Human All Exon v5 protocol from FFPE tumor tissue samples. The libraries were equimolar pooled. Each pool was sequenced on two lanes using the Illumina HiSeq 4000 System with 100 bp paired end sequencing. To ensure success quality control was verified with an Agilent 4200 TapeStation system of the received gDNA samples and during the library preparation. Results: Intermediate QC steps were taken throughout the protocol to monitor library preparation for sequencing, such as evaluation of DNA after fragmentation, analysis of adapter-ligated and amplified DNA, and lastly, qualification of the final library. The initial QC of incoming gDNA was determined based on the DNA integrity number (DIN). All samples had a low DNA integrity, what is usual for DNA extracted from FPPE material. Due to the low quality of the DNA material, a modified fragmentation protocol was used. Modification of the fragmentation enabled to obtain meaningful sequencing results. Conclusion: Quality control is an important part of NGS workflows, library preparation protocols recommend quantification and qualification of the DNA samples at various stages. The increasing sample throughput creates a need for automation especially in a core facility where many precious samples are proceeded with time pressure. The implementation of the automated electrophoresis system in the whole exome sequencing workflow enabled to increase the efficiency of the workflow and ensure good sequencing results.

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

Is capillary electrophoresis on microchip devices able to genotype uridine diphosphate glucuronosyltransferase 1A1 TATA-box polymorphisms?

In this commentary, we focused our attention on capillary electrophoresis. It achieves the efficient separation of molecular species by the application of high voltages to samples in solution. Actually, capillary electrophoresis can be performed on microchip devices, based on an automated and miniaturized electrophoresis system, based on lab-on-a-chip technology. By this technology it is possible to separate nucleic acid fragments (DNA or RNA) with respect to sizing accuracy and sizing resolution. Currently, two automated capillary electrophoresis on microchips devices are available: the Agilent 2100 Bioanalyzer and the Experion™ Automated Electrophoresis System. In this study, we evaluated if the CE is able to distinguish the three uridine diphosphate glucuronosyltransferase 1A1 TATA-box genotypes.

An audit of immunofixation requesting practices at a South African referral laboratory.

BackgroundIt is common practice in most chemical pathology laboratories for reflective immunofixation electrophoresis (IFE) to occur following the detection or suspicion of a paraprotein on serum protein electrophoresis (SPEP). The chemical pathology laboratory at Inkosi Albert Luthuli Central Hospital (IALCH) in Durban, South Africa, is currently the only non-private laboratory in the KwaZulu Natal province that performs SPEP analysis, with current practice requiring that the clinician request IFE following suggestion by the laboratory after a suspicious SPEP result.ObjectivesTo review the current process for IFE at IALCH in the context of reflective testing and to examine the use of the alpha-2-globulin/alpha-1-globulin ratio as a predictor of a positive IFE result.MethodsData for 1260 consecutive SPEP tests performed at the IALCH National Health Laboratory Service were collected between February and July 2011. SPEP and IFE were performed with a Sebia Hydrasys automated electrophoresis system. The alpha-2-globulin/alpha-1-globulin ratio was calculated using density of corresponding fractions on SPEP.ResultsAnalysis revealed that of the 1260 SPEPs performed during the analysis period, 304 IFEs were suggested by the reviewing pathologist. A total of 45 (15%) of the suggested IFEs were subsequently requested by the attending clinicians. Almost half (46.5%) (n = 20) of the suggested IFEs that were performed revealed the presence of a paraprotein. There was no statistically-significant difference between the alpha-2-globulin/alpha-1-globulin ratio for patients with positive or negative IFEs (p-value = 0.2).ConclusionsThis study reveals the need for reflective addition of IFE testing by the laboratory following suspicious findings on SPEP.

Serum proteins profile in Comisana lambs during the first month of life

Abstract. The aim of the present study was to measure the serum total proteins and the serum protein fractions (albumin, α1-, α2-, β-, and γ- globulins) of ten newborn lambs (Comisana breed) during the first 30 days of life in order to obtain useful information for neonatal care. From each animal, blood samples were collected via jugular venipuncture at the same hour (9.00) every 3 days for 30 days starting from birth (day 0). The concentrations of serum total proteins and albumin, α1-, α2-, β-, γ- globulins, and Albumin/Globulins (A/G) ratio were determined using an automated electrophoresis system. One-way repeated measures analysis of variance (ANOVA), followed by Bonferroni’s test, was used to determine significant differences between mean values of the studied parameters from the 1st to the 30th day of the experimental period. Data analysis of variance showed a statistical effect of days of life on total proteins, albumin, α1-globulins, β-globulins, γ-globulins and ratio A/G during the first 30 days of life (P<0.0001), while no statistical significant effect of days of life was observed on α2-globulins during the experimental period (P=0.27). The obtained results indicated that passive transfer status, determined from serum immunoglobulin concentration 24 hours after birth, is a significant source of variation in preweaning growth performance in dairy lambs. These finding make a contribution to the knowledge of physiological adaptation in lambs during the first 30 days of life and give useful information for the diagnosis and treatment of neonatal diseases.

Identification of candidate biomarkers using the Experion™ automated electrophoresis system in serum samples from ovarian cancer patients

Ovarian cancer is the most common cause of disease-related death in women globally. Detection of ovarian cancer using new biomarkers is necessary for early diagnosis. To date, there have been no obvious biomarkers for ovarian cancer detection in the incipient stage. In this study, we discovered potential diagnostic serological biomarkers for ovarian cancer using the Experion™ automated electrophoresis system. Sera from 14 healthy women and 84 ovarian cancer patients at stages I- IV were applied to the Experion to compare the protein expression levels. To examine the protein expression pattern of Experion data, proteins in the samples were resolved using 10 and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. The candidate biomarkers elevated in ovarian cancer were purified and determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. α-2-macroglobulin (173.7 kDa), ceruloplasmin (147 kDa), inter-α-trypsin inhibitor family heavy chain-related protein (126 kDa), C-1 inhibitor (115.2 kDa) and hemoglobin α/β (14.4 kDa were overexpressed in the ovarian cancer sera. This study documents a novel way to measure ovarian cancer or cancer-related proteins for biomarkers using the Experion assay system, which should be easily adaptable for high-throughput diagnosis to establish databases of ovarian cancer for clinical applications.

Predominant mucosal expression of 5-HT4(+h)receptor splice variants in pig stomach and colon

To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues. A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR. After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH. Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT.

Identification of M Protein From Filter Paper Using Serum Protein and Immunofixation Electrophoresis

Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are standard methods for detection and monitoring of monoclonal (M) proteins. However, these tests are rarely available in the remote areas, especially in developing countries. Transportation of fresh serum (FS) samples is also usually inconvenient. This study investigated M-protein identification using serum blot on filter paper (FP). SPE and IFE were performed on FS and FP specimens using the Sebia Hydrasys automated electrophoresis system. Statistical analyses were conducted to assess sample stability and agreement of FS vs FP. The FP method showed good agreement with the FS method. The r values for correlation of albumin levels-α(1), α(2), β, and γ (%)-between FP and FS samples in SPE were all more than 0.95 (P < .01). IFE displayed no significant difference between those 2 methods in the identification of M protein. The FP method demonstrated an accurate and reproducible alternative to FS for identification of M protein using SPE and IFE.

High-throughput sequencing identifies an NFIA/CBFA2T3 fusion gene in acute erythroid leukemia with t(1;16)(p31;q24)

High-throughput sequencing identifies an NFIA/CBFA2T3 fusion gene in acute erythroid leukemia with t(1;16)(p31;q24)

Detection of cryoglobulins in serum of Caspian miniature horse

Blood samples were collected from 20 healthy miniature Caspian horses at 37 °C. Isolation of cryoglobulin was performed based on a standard method in present study. Harvested sera were kept at 4 °C for two hours and then examined for cryoglubolin. Four serum samples containing precipitate Suspicious of containing cryoglobulin were selected. Subsequently serum protein electrophoresis was performed on normal serum samples and also on four serum samples containing precipitates using an automated electrophoresis system on cellulose acetate strips. In addition Ig isotypes detection (IgG, IgM and IgA) was performed on both precipitates and serum containing precipitates using single radio immunediffusion method (SRID). A narrow-based peak on gamma region of precipitate acetate cellulose strips was observed. Precipitate concentrations were strikingly higher than normal concentration of serum immuneglobulins. It can be suggested that cryoglobulin concentration in a proportion of Caspian miniature horse is higher than other equides may be in relation with animal susceptibility to neoplasias such as lymphoma and leukemia.

Increased Gamma Globulin Following Immunotherapy with Sipuleucel-T Is Associated with Antigen-Specific Antibody Responses

AbstractAbstract 1738Sipuleucel-T is an autologous cellular immunotherapy for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer. Sipuleucel-T consists of autologous peripheral blood mononuclear cells (PBMCs), including antigen presenting cells (APCs), that have been activated with a recombinant human protein, PAP-GM-CSF (prostatic acid phosphatase [PAP] linked to granulocyte-macrophage colony-stimulating factor [GM-CSF]). It is prepared from cells collected by leukaphereses at Weeks 0, 2, and 4 and infused approximately 3 days following each leukapheresis. In the IMPACT study, 512 patients were randomized (2:1) to sipuleucel-T or control (non-activated autologous PBMCs). Serum samples were collected at Baseline and Weeks 6, 14, and 26, and were analyzed for routine clinical chemistry tests in a comprehensive metabolic panel, which included total protein and albumin. In selected samples, serum protein electrophoresis (SPEP) and densitometry were performed using the Sebia automated electrophoresis system. We observed elevated total protein at Weeks 6 and 14 in sipuleucel-T patients only. At Week 6, 10.0% (28/279) of sipuleucel-T patients with a normal baseline total protein concentration had an elevated total protein concentration (upper limit of normal [ULN] = 7.9 to 8.5 g/dL, depending on age) compared with 0% (0/140) of control patients. At Week 14, 10.4% (19/183) of sipuleucel-T patients with a normal baseline total protein had an elevated total protein compared with 0% (0/86) of control patients. Albumin levels were not elevated at Week 6 following treatment (0.4% ½84 of sipuleucel-T patients with a normal baseline albumin concentration had increases in post-treatment serum albumin), indicating that the changes in total protein were due to elevations in serum globulin. In sipuleucel-T patients, there was a positive correlation between the maximum globulin protein elevations (total protein minus albumin) and the magnitude of antibody titers to PAP-GM-CSF at Week 6 (n = 126; P = 0.001) and Week 14 (n = 85; P = 0.002). Samples from sipuleucel-T patients for whom both a baseline and post-treatment sample were available and who fell within the upper 10th percentile of the total protein change (n = 13) were evaluated by SPEP. All of these patients had increased gamma globulin compared with baseline. Nine patients (69.2%) developed gamma globulin above the upper limit of normal (i.e., > 1.5 g/dL) which was not present at baseline. A correlation between gamma globulin elevations and antibody titers to PAP-GM-CSF in these patients (n = 9) was observed at Week 6 (r = 0.664, P = 0.051). These data demonstrate that elevated gamma globulin levels can develop following treatment with sipuleucel-T and were associated with the development of an antigen-specific humoral response. Disclosures:Sims:Dendreon Corp.: Employment. dela Rosa:Dendreon Corp.: Employment. Lin:Dendreon Corp.: Employment. Sheikh:Dendreon Corp.: Employment.

Phytogeographical origin of Madeiran common beans based on phaseolin patterns

The objective of this work was to determine the geographic origin of the Madeiran common bean (Phaseolus vulgaris) gene pool. Phaseolin patterns of 50 accessions representing the diversity of common bean collected in Madeira, Portugal, and conserved in the ISOPlexis Germplasm Bank, were analysed using the Experion automated electrophoresis system, based on lab-on-a-chip technology. Five common bean standard varieties with typical phaseolin patterns were used to determine the phytogeographical origin of the Madeiran common bean accessions. Ninety two percent of the accessions exhibited a phaseolin pattern consistent with the one of common bean types belonging to the Andean gene pool, while the origin of the remaining 8% of the accessions was indistinguishable. The application of a similarity coefficient of 85%, based on Pearson correlations, increases the number of accessions with uncertain pattern. The analytical approach used permitted the determination of the origin of the common bean gene pool, which is Andean in 98% of the cases, and clustering of the observed variability among the Madeiran common beans.

Microarray-based Transcriptional and Epigenetic Profiling of Matrix Metalloproteinases, Collagens, and Related Genes in Cancer

Epigenetic parameters (DNA methylation, histone modifications, and miRNAs) play a significant role in cancer. To identify the common epigenetic signatures of both the individual matrix metalloproteinases (MMPs) and the additional genes, the function of which is also linked to proteolysis, migration, and tumorigenesis, we performed epigenetic profiling of 486 selected genes in unrelated non-migratory MCF-7 breast carcinoma and highly migratory U251 glioma cells. Genome-wide transcriptional profiling, quantitative reverse transcription-PCR, and microRNA analyses were used to support the results of our epigenetic studies. Transcriptional silencing in both glioma and breast carcinoma cells predominantly involved the repressive histone H3 Lys-27 trimethylation (H3K27me3) mark. In turn, epigenetic stimulation was primarily performed through a gain in the histone H3 Lys-4 dimethylation (H3K4me2) and H3 hyperacetylation and by a global reduction of H3K27me3. Inactive pro-invasive genes in MCF-7 cells but not in U251 cells frequently exhibited a stem cell-like bivalent mark (enrichment in both H3K27me3 and H3K4me2), a characteristic of developmental genes. In contrast with other MMPs, MMP-8 was epigenetically silenced in both cell types, thus providing evidence for the strict epigenetic control of this anti-tumorigenic proteinase in cancer. Epigenetic stimulation of multiple collagen genes observed in cultured glioma cells was then directly confirmed using orthotopic xenografts and tumor specimens. We suggest that the epigenetic mechanisms allow gliomas to deposit an invasion-promoting collagen-enriched matrix and then to use this matrix to accomplish their rapid migration through the brain tissue.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression. siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks. These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology. Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages. To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis. The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa). The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells. We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression. siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks. These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology. Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages. To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis. The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependant gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa). The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells. We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

213 ANALYSIS OF STEROID HORMONE RECEPTOR GENE EXPRESSION IN THE BOVINE OVARY AFTER LASER-CAPTURE MICRODISSECTION

Developmental processes in complex organs like the ovary are difficult to study in terms of a biochemical and molecular biological analysis. Laser-assisted microdissection allows the efficient and precise capture of single cells or groups of cells of an organ within the context of time and space and permits their subsequent molecular characterization. Together with real-time PCR techniques, it is now feasible to study gene expression in defined cell populations of complex tissues, but it is essential to create standards optimized for fixation, preparation, and isolation of RNA, reverse transcription reaction, and real-time PCR protocol for every tissue of interest. The aim of our study was to develop protocols for a precise analysis of estrogen receptor alpha (ER-α) and progesterone receptor (PR) in defined compartments of the ovary (granulosa cells, theca interna cells, zona vasculosa, and zona parenchymatosa of the stroma). Additionally, the receptor proteins were localized by immunohistochemistry. A special focus was put on the question of how formalin fixation and paraffin embedding influences the quality of the isolated RNA from microdissected material, which was used for quantitative reverse transcription-PCR (qRT-PCR). Quality and quantity of total RNA extracted from formalin-fixed, paraffin-embedded (FFPE) sections and from material immersed in RNAlater® (Ambion, Foster City, CA, USA) was checked using an Experion automated electrophoresis system (Bio-Rad, Munich, Germany). The RNA quality indicator for microdissected material was between 6 and 7, and for RNAlater® material was 9 or better. Online qRT-PCR using the iCycler SYBR GreenTM protocol (Bio-Rad) was performed in a 96-well plate. Primer pairs were chosen to generate PCR products between 100 bp (ER-α) and 140 bp (PR), as RNA recovered from FFPE-laser microdissected material was expected to be considerably fragmented. Using GenEx software (BioEPS, Freisling, Germany), we showed that the expression of mRNA for PR was much stronger in the theca interna than in the 3 other compartments. Estrogen receptor alpha, on the other hand, was nearly exclusively expressed in the zona parenchymatosa and zona vasculosa of the stroma. Our results show that cells obtained after laser microdissection from FFPE ovarian material can be successfully used for subsequent real-time PCR, despite the fact the RNA quality indicator number of the isolated RNA was usually comparatively low. The data of our immunohistochemical analysis support the expression data of our RNA studies. In conclusion, laser-capture microdissection in combination with quantitative PCR is a reproducible and reliable technique for quantification of a small number of cells from FFPE material. We gratefully acknowledge the continuous support by the DFG-Graduiertenkolleg 1029 and the BMBF (ARG 08/013).