Comparison of antigen detection and quantitative PCR in the detection of chlamydial infection in koalas (Phascolarctos cinereus)

The gold standard method for detecting chlamydial infection in domestic and wild animals is PCR, but the technique is not suited to testing animals in the field when a rapid diagnosis is frequently required. The objective of this study was to compare the results of a commercially available enzyme immunoassay test for Chlamydia against a quantitative Chlamydia pecorum-specific PCR performed on swabs collected from the conjunctival sac, nasal cavity and urogenital sinuses of naturally infected koalas (Phascolarctos cinereus). The level of agreement for positive results between the two assays was low (43.2%). The immunoassay detection cut-off was determined as approximately 400 C. pecorum copies, indicating that the test was sufficiently sensitive to be used for the rapid diagnosis of active chlamydial infections.

Cohesive zone modelling of mineralized collagen fibril arrays in bending

Hard biological materials such as bone possess superior material properties of high stiffness and toughness. Two unique characteristics of bone microstructure are a large aspect ratio of mineralized collagen fibrils (MCF), and an extremely thin and large area of extrafibrillar protein matrix located between the MCF. The objective of this study is to investigate the effects of: (1) MCF aspect ratio, and (2) energy dissipation in extrafibrillar protein matrix on the mechanical behaviour of MCF arrays. In this study, notched specimens of MCF arrays in extrafibrillar protein matrix are subjected to bending. Cohesive zone model was implemented to simulate the failure of extrafibrillar protein matrix. The study reveals that the MCF array with a higher MCF aspect ratio and the MCF array with a higher protein energy dissipation in the interface direction are able to sustain a higher bending force and dissipate higher energy.

A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

Background Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation. Methods The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing. Results The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). Conclusions Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.

Development of an eight gene expression profile implicating human breast tumours of all grade

The goal of improving systemic treatment of breast cancers is to evolve from treating every patient with non-specific cytotoxic chemotherapy/hormonal therapy, to a more individually-tailored direct treatment. Although anatomic staging and histological grade are important prognostic factors, they often fail to predict the clinical course of this disease. This study aimed to develop a gene expression profile associated with breast cancers of differing grades. We extracted mRNA from FFPE archival breast IDC tissue samples (Grades I–III), including benign tumours. Affymetrix GeneChip� Human Genome U133 Plus 2.0 Arrays were used to determine gene expression profiles and validated by Q-PCR. IHC was used to detect the AXIN2 protein in all tissues. From the array data, an independent group t-test revealed that 178 genes were significantly (P B 0.01) differentially expressed between three grades of malignant breast tumours when compared to benign tissues. From these results, eight genes were significantly differentially expressed in more than one comparison group and are involved in processes implicated in breast cancer development and/or progression. The two most implicated candidates genes were CLD10 and ESPTI1 as their gene expression profile from the microarray analysis was replicated in Q-PCR analyses of the original tumour samples as well as in an extended population. The IHC revealed a significant association between AXIN2 protein expression and ER status. It is readily acknowledged and established that significant differences exist in gene expression between different cancer grades. Expansion of this approach may lead to an improved ability to discriminate between cancer grade and other pathological factors.

Semaphorin–plexin signalling genes associated with human breast tumourigenesis

Introduction Gene expression profiling has enabled us to demonstrate the heterogeneity of breast cancers. The potential of a tumour to grow and metastasise is partly dependant on its ability to initiate angiogenesis or growth and remodelling of new blood vessels, usually from a pre-existing vascular network, to ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells along with access to the systemic circulation. Cell–cell signalling of semaphorin ligands through interaction with their plexin receptors is important for the homeostasis and morphogenesis of many tissues and has been widely studied for a role in neural connectivity, cancer, cell migration and immune responses. This study investigated the role of four semaphorin/plexin signalling genes in human breast cancers in vivo and in vitro. Materials and methods mRNA was extracted from formalin fixed paraffin embedded archival breast invasive ductal carcinoma tissue samples of progressive grades (grades I–III) and compared to tissue from benign tumours. Gene expression profiles were determined by microarray using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays and validated by Q-PCR using a Corbett RotorGene 6000. Following validation, the gene expression profile of the identified targets was correlated with those of the human breast cancer cell lines MCF-7 and MDA-MD-231. Results The array data revealed that 888 genes were found to be significantly (p ≤ 0.05) differentially expressed between grades I and II tumours and 563 genes between grade III and benign tumours. From these genes, we identified four genes involved in semaphorin–plexin signalling including SEMA4D which has previously been identified as being involved in increased angiogenesis in breast cancers, and three other genes, SEMA4F, PLXNA2 and PLXNA3, which in the literature were associated with tumourigenesis, but not directly in breast tumourigenesis. The microarray analysis revealed that SEMA4D was significantly (P = 0.0347) down-regulated in the grade III tumours compared to benign tumours; SEMA4F, was significantly (P = 0.0159) down-regulated between grades I and II tumours; PLXNA2 was significantly (P = 0.036) down-regulated between grade III and benign tumours and PLXNA3 significantly (P = 0.042) up-regulated between grades I and II tumours. Gene expression of SEMA4D was validated using Q-PCR, demonstrating the same expression profile in both data sets. When the sample set was increased to incorporate more cases, SEMA4D continued to follow the same expression profile, including statistical significance for the differences observed and small standard deviations. In vitro the same pattern was present where expression for SEMA4D was significantly higher in MDA-MB-231 cells when compared to MCF-7 cells. The expression of SEMA4F, PLXNA2 and PLXNA3 could not be validated using Q-PCR, however in vitro analysis of these three genes revealed that both SEMA4F and PLXNA3 followed the microarray trend in expression, although they did not reach significance. In contrast, PLXNA2 demonstrated statistical significance and was in concordance with the literature. Discussion We, and others, have proposed SEMA4D to be a gene with a potentially protective effect in benign tumours that contributes to tumour growth and metastatic suppression. Previous data supports a role for SEMA4F as a tumour suppressor in the peripheral nervous system but our data seems to indicate that the gene is involved in tumour progression in breast cancer. Our in vitro analysis of PLXNA2 revealed that the gene has higher expression in more aggressive breast cancer cell types. Finally, our in vitro analysis on PLXNA3 also suggest that this gene may have some form of growth suppressive role in breast cancer, in addition to a similar role for the gene previously reported in ovarian cancer. From the data obtained in this study, SEMA4D may have a role in more aggressive and potentially metastatic breast tumours. Conclusions Semaphorins and their receptors, the plexins, have been implicated in numerous aspects of neural development, however their expression in many other epithelial tissues suggests that the semaphorin–plexin signalling system also contributes to blood vessel growth and development. These findings warrant further investigation of the role of semaphorins and plexins and their role in normal and tumour-induced angiogenesis in vivo and in vitro. This may represent a new front of attack in anti-angiogenic therapies of breast and other cancers.

Matrix metalloproteinase localisation by in situ-RT-PCR in archival human breast biopsy material

Utilising archival human breast cancer biopsy material we examined the stromal/epithelial interactions of several matrix metalloproteinases (MMPs) using in situ-RT-PCR (IS-RT-PCR). In breast cancer, the stromal/epithelial interactions that occur, and the site of production of these proteases, are central to understanding their role in invasive and metastatic processes. We examined MT1-MMP (MMP-14, membrane type-1-MMP), MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) for their localisation profile in progressive breast cancer biopsy material (poorly differentiated invasive breast carcinoma (PDIBC), invasive breast carcinomas (IBC) and lymph node metastases (LNM)). Expression of MT1-MMP, MMP-1 and MMP-3 was observed in both the tumour epithelial and surrounding stromal cells in most tissue sections examined. MT1-MMP expression was predominantly localised to the tumour component in the pre-invasive lesions. MMP-1 gene expression was relatively well distributed between both tissue compartments, while MMP-3 demonstrated highest expression levels in the stromal tissue surrounding the epithelial tumour cells. The results demonstrate the ability to distinguish compartmental gene expression profiles using IS-RT-PCR. Further, we suggest a role for MT1-MMP in early tumour progression, expression of MMP-1 during metastasis and focal expression pattern of MMP-3 in areas of expansion. These expression profiles may provide markers for early breast cancer diagnoses and present potential therapeutic targets.

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

Background Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. Methods We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. Results In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. Conclusion We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

An examination of MS candidate genes identified as differentially regulated in multiple sclerosis plaque tissue, using absolute and comparative real-time Q-PCR analysis

In our laboratory, we have developed methods in real-time detection and quantitative-polymerase chain reaction (Q-PCR) to analyse the relative levels of gene expression in post mortem brain tissues. We have then applied this method to examine differences in gene activity between normal white matter (NWM) and plaque tissue from multiple sclerosis (MS) patients. Genes were selected based on their association with pathology and through identification by previously conducted global gene expression analysis. Plaque tissue was obtained from secondary progressive (SP) patients displaying chronic active, as well as acute pathologies; while NWM from the same location was obtained from age- and sex-matched controls (normal patients). In this study, we used both SYBR Green I supplementation and commercially available mixes to assess both comparative and absolute levels of gene activity. The results of both methods compared favourably for four of the five genes examined (P < 0.05, Pearsons), while differences in gene expression between chronic active and acute pathologies were also identified. For example, a >50-fold increase in osteopontin (Spp1) and inositol 1-4-5 phosphate 3 kinase B (Itpkb) levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P < 0.05, unpaired t test). By contrast, there was no significant difference in the levels of the MS marker and calcium-dependent protease (Calpain, Capns1) in MS plaque tissue. In summary, Q-PCR analysis using SYBR Green I has allowed us to economically obtain what may be clinically significant information from small amounts of the CNS, providing an opportunity for further clinical investigations.

The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis

In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A(1), A(2A), A(2B) and A(3), and estrogen receptors (ER) alpha and beta. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA(2A) (>4-fold down) and consistent (>2-fold) down-regulation of ADORA(1), ADORA(3), and ER-beta, following ovariectomy. No change was found in ADORA(2B) or ER-alpha. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain.

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

β-Actin—an unsuitable internal control for RT-PCR

Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which β-actin is also regulated, the mRNA for β-actin remains a widely used housekeeping gene internal control. Utilizing differential reverse transcriptase-polymerase chain reaction (RT-PCR), we report here the dose-dependent inhibition of β-actin by matrigel. This was detected by comparison to the very moderate inhibition of the target gene, membrane type-1 matrix metalloproteinase (MT1-MMP), with results independently confirmed by similar findings on MT1-MMP expression using competitive RT-PCR. Furthermore, RT-PCR of the housekeeping gene 18 Svedberg Units (S) rRNA demonstrated excellent consistency, reproducibility and non-regulation by a matrigel treatment. We conclude that β-actin is highly regulated by matrigel and therefore unsuitable as an internal control in this treatment. Hence, these findings suggest that researchers have a responsibility to ensure that the housekeeping gene of choice is not regulated in their specific application, as such regulation may dramatically affect the accuracy of their results. This study reinforces the necessity for minimally regulated housekeeping genes such as 18S rRNA, and the superiority of competitive templates as internal controls for quantitative applications of RT-PCR.

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human β-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene β-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.

Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

Molecular genetic analyses of RFLPs for PCR-amplified growth hormone gene, renal kallikrein gene and atrial natriuretic factor gene in essential hypertension

The present study examined polymorphisms of genes that might be involved in the onset of essential hypertension (HT). These included the (i) growth hormone gene (GH1), whose locus has recently been linked to elevated blood pressure (BP) in the stroke-prone SHR, although recent sib-pair analysis of a polymorphism near the human chorionic somatomammotropin gene (a member of the GH cluster) was unable to show linkage with HT; (ii) renal kallikrein gene (KLK1); and (iii) atrial natriuretic factor gene (ANF), where a primary defect in production or activity of kallikrein or ANF could cause NaCl retention and vasoconstriction. Association analyses were conducted to compare restriction fragment length polymorphisms (RFLPs) of each gene in 85 HT and 95 normotensive (NT) Caucasian subjects whose parents had a similar BP status at age ≥50 years. The frequency of the minor allele of (i) a RsaI RFLP in the promoter of GH1, amplified from leukocyte DNA by the polymerase chain reaction, was 0.15 in the HT group and 0.14 in the NT group (χ1=0.34, P=0.55); (ii) a TaqI RFLP for KLK1 was 0.035 in the HT group and 0.015 in the NT group (χ2=1.5, P=0.21); and (iii) a XhoI RFLP for ANF was 0.50 in HTs and 0.46 in NTs (χ2=0.20, P=0.65). Studies of HT pedigrees found one family in which the ANF locus and HT were not linked, owing to an obligate recombinant. The present data thus provide no evidence for involvement of the growth hormone, renal kallikrein, nor ANF gene in the causation of essential hypertension.

Design, construction, and analysis of cell line arrays and tissue microarrays for gene expression analysis

Cell line array (CMA) and tissue microarray (TMA) technologies are high-throughput methods for analysing both the abundance and distribution of gene expression in a panel of cell lines or multiple tissue specimens in an efficient and cost-effective manner. The process is based on Kononen's method of extracting a cylindrical core of paraffin-embedded donor tissue and inserting it into a recipient paraffin block. Donor tissue from surgically resected paraffin-embedded tissue blocks, frozen needle biopsies or cell line pellets can all be arrayed in the recipient block. The representative area of interest is identified and circled on a haematoxylin and eosin (H&E)-stained section of the donor block. Using a predesigned map showing a precise spacing pattern, a high density array of up to 1,000 cores of cell pellets and/or donor tissue can be embedded into the recipient block using a tissue arrayer from Beecher Instruments. Depending on the depth of the cell line/tissue removed from the donor block 100-300 consecutive sections can be cut from each CMA/TMA block. Sections can be stained for in situ detection of protein, DNA or RNA targets using immunohistochemistry (IHC), fluorescent in situ hybridisation (FISH) or mRNA in situ hybridisation (RNA-ISH), respectively. This chapter provides detailed methods for CMA/TMA design, construction and analysis with in-depth notes on all technical aspects including tips to deal with common pitfalls the user may encounter. © Springer Science+Business Media, LLC 2011.