Computational thermal analysis of a continuous flow micro Polymerase Chain Reaction (PCR) chip

The first continuous flow micro PCR introduced in 1998 has attracted considerable attention for the past several years because of its ability to amplify DNA at much faster rate than the conventional PCR and micro chamber PCR method. The amplification is obtained by moving the sample through 3 different fixed temperature zones. In this paper, the thermal behavior of a continuous flow PCR chip is studied using commercially available finite element software. We study the temperature uniformity and temperature gradient on the chip’s top surface, the cover plate and the interface of the two layers. The material for the chip body and cover plate is glass. The duration for the PCR chip to achieve equilibrium temperature is also studied.

Optimisation and analysis of three polymerase chain reaction (PCR) systems for the detection of environmentally significant microorganisms

Conventional assessments of water quality are based on the determination of bacterial indicator numbers. The detection of an indicator organism only provides presumptive evidence of the presence of harmful organisms By using polymerase chain reaction (PCR) to monitor water quality, indicators and pathogenic organisms can be specifically detected with a high sensitivity. The research illustrates the importance of optimisation and the maintenance of reaction efficiency in the accuracy and precision of the PCR.

Effectiveness of alternative organic solvents in field preservation of whole barnacles for PCR analyses

There are few reports of non-cryogenic preservation methods for marine invertebrates, so potable alcohol and acetone-based nail varnish remover (NVR) are for the first time evaluated against absolute ethanol as short-term preservatives of whole barnacles. Performance of ethanol and NVR-preserved material was comparable, but potable alcohol was significantly worse. These results are of practical importance for fieldwork in remote areas where laboratory chemicals are unattainable but potable alcohol or NVR are locally available. Of these, acetone-based NVR would be the solvent of preference.

Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice: Real-time PCR quantitation

Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial β-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan® real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitate metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 μm segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n=48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r 2=0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called `vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.