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Thank you Dr Aparna Jayachandran from Greenslopes Private Hospital, for a great review on our ISOLATE II RNA Mini Kit.
"We have been utilizing ISOLATE II RNA Mini Kit from Bioline and have found that the RNA yield, concentrations, and purity have been consistently good. From 40,000 liver cancer (mouse and human) cells we are able to get 200-600ng/ul yield of RNA."
Click the link to learn more about >> ISOLATE II RNA Mini Kit
Thank you Rebecca Traub from University of Melbourne, for a great review on our ISOLATE Fecal DNA Kit
"Aim: To compare the sensitivity of two commercial faecal DNA extraction kits for the detection and quantification of human hookworms (Ancylostoma ceylanicum, Ancylostoma duodenale and Necator americanus) eggs in faeces.
Methods: Ten individual human faecal samples positive for hookworm eggs were each extracted with two commercial kits, the Powersoil DNA Isolation Kit (MoBio) and the ISOLATE Fecal DNA Kit (Bioline) according to manufacturer's instructions. DNA was eluted to 100 ul for each.
DNA was subjected to multiplex Taq-man based multiplex qPCR (unpublished) targeting the internal transcribed spacer regions of the three human hookworms and an internal control (EHV4).
Results: Both kits performed equivocally in positively detecting at least one species of hookworm in all ten samples. The ISOLATE Fecal DNA Kit produced on average 0.5 Ct lower threshold cycles for samples compared to the Powersoil DNA Isolation Kit. The time taken to extract 10 samples was significantly less for the Bioline product.
Conclusion: The Powersoil DNA Isolation Kit and the ISOLATE Fecal DNA Kit are comparable in terms of sensitivity for detecting and quantifying DNA of hookworms from human faecal samples. The Bioline kit was less labour intensive compared to the Mo Bio kit and is more competitively priced."
Click the link to view the data >> Hookworm Multiplex Kit Data
Click the link to learn more about >> ISOLATE Fecal DNA Kit
Thank you Jonathan Ferrand from Hudson Institute of Medical Research, VIC Australia, for a great review on our MyTaq™ Red Mix
"We have been using the MyTaq™ Red Mix for the past two years in our routine PCRs for mice genotyping, cloning/amplicon validation and standard curve preparation for our RTqPCR. This product is very convenient with gel loading buffer included, and in our hands work as well as competitor products with as low as 5ul of Mix per reaction (10ul final volume). The competitive pricing makes it a very attractive alternative for routine PCRs."
Click the link to learn more about >> MyTaq™ Red Mix
Initially, the RT step should be performed as specified in the supplier protocol. However, the length and the temperature of the RT step can be optimized to increase the efficiency of the reverse transcriptase. The reverse transcriptase should be tested across a range of RNA concentrations to ensure assay linearity.
The field of molecular diagnostics has seen much growth in the clinical setting, providing rapid and sensitive approaches for the detection and monitoring of a wide range of human ailments. There is very real potential for molecular diagnostics to revolutionise patient care, offering tools that go further than simple characterisation of disease, reaching into the domain of characterising the patient.
Bioline offers a range of products and services to support the research that contributes towards development of diagnostic products and testing services. With ISO13485 manufacturing standards, each reagent represents the level of quality required when developing tests for the clinical market. In addition, our custom assay development services can provide the edge required for optimal performance and expedient development of your next molecular diagnostic assay.
What is Molecular Diagnostics?
Diagnostic or clinical pathology plays an essential role in patient care – providing physicians with the specific information required to identify and treat the very broad range of ailments presented across primary doctor or hospital practice settings. Molecular diagnostic approaches utilise nucleic acid detection techniques to analyse target DNA or RNA from an affected individual. Molecular-based tests can cover a range of clinical conditions from inherited genetic disease, through the full range of cancers, infectious disease agents, drug-dose or treatment response scenarios (pharmacogenomics), and even personalised treatment and prognostic investigations based upon individual genetic make-up (personalised medicine). Results from molecular diagnostic tests are used in conjunction with the presented symptoms and clinical expertise of the serving physician to better understand disease aetiology, pathogenesis, diagnosis and prognosis.
Technology common within a research environment is not always readily adopted in a diagnostic setting. Diagnostic tests must demonstrate clinical utility, while at the same time adhere to strict quality requirements for reproducibility, along with appropriate sensitivity and specificity performance. Although molecular biology has been a field of study for over 50 years, the integration of molecular diagnostics into pathological fields has been variable. While clinical genetics has become almost entirely molecular-based, traditional morphological analysis, chemical analysis, and immunohistochemistry will always have a place in many areas.
Molecular diagnostics covers a range of techniques from fluorescent in-situ hybridisation (FISH), DNA-chip technology, mass spectrometry, as well as nucleic acid amplification tests (NAATs). The revolution in molecular diagnostics came with the adoption of the Polymerase Chain Reaction (PCR) and the completion of the Human Genome Project. Both scientific milestones have served to expand the usefulness and range of applicability for molecular diagnostic approaches. Data from the Human Genome Project have opened up many possible targets for detection, prevention and/or treatment of disease. PCR is now the most commonly used molecular diagnostic tool, offering a very sensitive and rapid approach for the detection, identification, and quantification of specific DNA or RNA targets. More recently Real-Time PCR, utilising fluorescent dye detection, has streamlined the use of NAATs - improving quantification applications, turn-around times, and significantly reducing the risk of carry-over contamination.
Molecular Diagnostics – Current Applications
From individual buffers and enzymes, sample preparation reagents and Real-Time PCR Kits, right through to fully designed PCR assays, Bioline molecular reagents and services are quality manufactured for your nucleic-acid based testing (NAAT) needs. Molecular diagnostic approaches routinely use NAATs across a range of applications, some of which are summarised here.
Although traditional culture methods are still invaluable for pathogen identification and investigation of specific treatment sensitivities (such as antibiotic resistance screening), molecular diagnostics, in particular PCR and Real-Time PCR NAATs, are routinely used for a range of infectious disease testing. The very sensitive and rapid approach of molecular tests support clinical decisions for diagnosis and treatment of a range of bacterial and viral infections, allowing for very specific discrimination of individual target strains, as well as accurate quantification for monitoring organism load across a treatment regime. Molecular diagnostics are also routinely used for screening purposes - from sexually transmitted diseases right through to screening blood products for potential pathogens.
Mutations in a DNA sequence can change the resulting translated protein with the potential for ongoing effects on the subsequent phenotype or active characteristics of an organism. The most common genetic variants are in the form of single nucleotide polymorphisms (SNPs), and these can confer a broad range of characteristics such as antibiotic resistance in bacteria, or a change in likely response to certain drug treatments, thus there is a range of SNP molecular diagnostic tests in routine practice today. SNPs can be detected using a range of methods including sequence-specific PCR, Dual-labelled hybridisation probe discrimination, and post-PCR analyses, such as amplicon melting or restriction fragment polymorphism approaches. As deep sequencing technologies improve and more genome data is gathered across a range of organisms, the utility and application of SNP testing will only increase.
Biomarkers are objectively measured characteristics, used as indicators to monitor biological or pathological processes, as well as pharmacological responses to treatment or therapy. Biomarkers can cover a range of substances and molecules - such as whole cells, enzymes or hormones. Specific genes or gene products are often targeted for biomarker applications, particularly in the field of drug development, allowing for stratification of a population based on genotype or presence of RNA marker. Although molecular biomarkers show real promise in research and development settings, their routine use in a clinical setting are often hampered by the logistical challenges of standardised measurement processes, along with requirements for robust validation of analytical procedures, and heavy data requirements for clinical validity.
Emerging trends in personalised medicine
The recent boom in deep sequencing technologies and computational biology has expanded biological study into the era of “omics” – such as genomics and proteomics – with simultaneous analysis of hundreds or thousands of genes or proteins. The most direct and current application of this high-volume, high-throughput approach is in the area of array-based technologies, where hundreds of targets can be tested in parallel to gain a large-scale gene expression profile. A clinical setting example of this approach is the array test for detection of mutations or polymorphisms in the genes of the cytochrome P450 system, responsible for metabolism of a range of medications. However, in general, the transition of this rise in genetic information has been slow to cross into the molecular diagnostic laboratory. The challenge lies in understanding the vast amount of data and translating that into clinically useful information. True personalised medicine is the ultimate goal – identifying the individual differences that lead to disease susceptibility, treatment response differences, ultimate disease progression and therapeutic outcomes, and research is continuing toward that end.
Molecular Diagnostics – Maintaining the Gold Standard
In addition to demonstrating the clinical utility and scientific validity of a test, in order for it to be applied in a clinical or diagnostic setting, a number of performance characteristics need to be established, including precision, accuracy, analytical sensitivity and specificity, along with a range of quality determinants covering manufacturing processes, reproducibility, and laboratory staff training requirements. These processes are monitored by local governing bodies, and molecular diagnostic laboratories are required to undergo routine testing and certification updates to maintain their status for reporting clinical results. For these reasons alone, introduction of new technologies or tests is a slow process in molecular diagnostics, and examples of erroneous results in the form of false positive, false negative, or those of no clear relevance to disease state, continue to be reported across the field. Although PCR is now widely adopted in molecular diagnostics, this technology holds its own inherent caveats, including finding appropriate reference sequences, or endogenous control genes for accurate detection or gene expression analysis. Finding a trusted source for core reagents that offer reliability, reproducibility and performance standards that support the demands of clinical specificity and sensitivity is always key to a successful application in a molecular diagnostic setting, and with ISO13485 manufacturing standards and a commitment to quality assay design services, Bioline is a well-placed choice to support your laboratory quality standards.
The best reward for us is hearing achievements through Bioline products, Kristina from West Virginia University speaks about why she gives Accuzyme Mix the “Thumbs Up”
Well done Jesse for working so closely and showing true commitment to his customers!