Agricultural Biotechnology and Environmental Research
Agriculture and the very environments we exist within are undeniably important and there is an interplay between them. Ecosystems not only provide vital life resources such as clean air and water, they also provide healthy land upon which to grow crops or farm livestock – about half of the ‘useable’ land across the globe is currently pastoral or employed in intensive agriculture. Research to support better understanding and management of agricultural practices, in addition to understanding and maintaining the wide variety of global ecosystems, is critical to a sustainable future for our growing population. There are a range of molecular tools available from Bioline to assist investigations within agricultural or environmental systems, whether through PCR, real-time PCR, or sequencing. Bioline also offers sample-to-answer workflows specially formulated for plant samples, streamlining and simplifying your laboratory time and maximizing result quality from these often difficult sample types.
Agriculture is a broad term encompassing the production, processing and distribution of crops, livestock, feed and other products that support humanity. In addition to traditional crop farming, many other industries fall under the agricultural banner including dairy, livestock, fisheries and forestry. Agriculture is often linked to definitions of civilization and there is evidence of plant breeding and animal domestication going back tens of thousands of years in many cultures.
From very early on, farmers recognized that desirable traits could be enhanced through breeding strategies – improving yields, disease resistance and other characteristics. It was through the study of plant breeding that Mendel identified the genotype-phenotype connection and described the fundamental laws of inheritance, often described as the founding of genetic science. While conventional breeding techniques have proven effective, results can often take multiple generations to manifest and sometimes it is difficult to identify a desired trait within existing populations. With a growing understanding of the genetic sequences and make-up of selected species, a more targeted approach looks at total genomics - linking complex phenotypic traits to the genetic architecture and underlying molecular mechanisms to better instruct cross breeding and back breeding efforts.
Genomics Assisted Breeding
Genomics assisted breeding programs greatly enhance selection efficiency through the construction of comprehensive genetic maps and establishment of marker-trait associations, directing molecular breeding programs for more precise outcomes. Molecular markers such as single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) or microsatellites are used to genotype segregating populations. With ongoing advances and applications of next generation sequencing (NGS), understanding of the genetic diversity within breeding material is increasing at a rapid pace, helping to improve knowledge of so-called ‘orphan’ crops and integrate the various core components of molecular breeding programs. Bioline offers a full range of sample preparation, cloning, sequencing and PCR reagents to support molecular breeding research and applications in all areas of agricultural science.
Breeding and Genetically Modified Organisms
The latest approach to breeding and agricultural crop improvement uses recombinant DNA techniques to genetically modify plants and animals. Individual genes that confer a desired trait, such as insect resistance or herbicide tolerance, are selected and transferred into the desired organism. Unlike conventional breeding, genetic modification (GM) can occur between completely unrelated species, thus opening access to a broader range of traits. GM breeding holds promise to support the future demands of a growing population and a changing climate, using traits such as drought and frost resistance, elevated iron or vitamin levels, or faster growth and maturation periods.
In order for transgenic breeding to be successful, it is necessary to have full understanding of the stability of the introduced gene across generations, as well as potential intergenic, intragenic or other interactions within the genome and within the environment where the crop or organism is being introduced. Nations across the Americas, Europe and Asia have embraced GM technology for many core crops. The US has granted approval for use of over 70 different GM varieties, with over 90% of cotton and 80% of corn across the US planted with GM seed. Approval has also been granted for the first use of GM fish – a salmon requiring half the usual time to grow to market size. Controversies surrounding GM technology mainly focus on the broader potential impacts of GM organisms on the surrounding ecosystems. Unknown future stability of engineered traits, the ability of organisms to transfer genes and breed naturally outside of agricultural constraints, competition with native plants and animals and other unintended and poorly understood outcomes, have seen some governments and organisations show caution towards the technology.
Our global environment and the individual ecosystems within are a complex interplay of a multitude of species and ecological processes and our understanding of all of that is limited at best. Our very existence relies on the continuation of healthy biological and ecological global and local systems and much of today’s research looks at how to define a ‘healthy’ system, how to monitor, measure and manage these systems, in order to maximize sustainable interaction. Apart from the intrinsic value of a healthy environment, essentials such as functional water cycles, soil formation and climate regulation, we also gain value from environmental services such as food, raw materials and fuels. Yet our agricultural and harvesting practices reduce the ability of ecosystems to continue to provide these services, thus effective and sustainable management practices and policies are key to our continued survival and sustainable future.
Although any measurable parameter of an ecosystem will offer valuable information, it is impossible to assess every aspect of complex natural systems, thus researchers look to define ‘indicators’ – biologically relevant measurements or surrogate organisms, that can represent or quantify key measures of an environment and offer insight into potential stress and or resilience of an environment. While typically a small group of key indicator organisms are studied in depth in order to assess ecosystem health, it is well recognized that it is the overall biodiversity within an ecosystem that offers the best opportunity for long-term stability, productivity and maximum functionality. This current epoch has been defined as the Anthropocene, a time when humans are a persistent and driving force of extinction – we are losing species 1,000 times faster than at any other point in history. The true impact of such events is yet to be seen and a sustainable relationship between humans and the global environment is not yet secure. Research efforts continue in an attempt to better define our current and future ecological state, using a range of methods including molecular characterisations of extant diversity and Bioline’s extensive range of molecular tools are well placed to support.
Methods for Assessing Biodiversity
Biodiversity can be defined on many different levels: from macro-level ecosystem diversity; species-level diversity of plants, animals or microorganisms; right down to the genetic diversity within individual populations. While species diversity is the most commonly used method for defining or measuring biodiversity in a given ecosystem, it is, in practice, a difficult variable to measure both in terms of appropriate sample size, spatial scale and method of identification, as well as in the very definition of diversity, be it richness, evenness, differentiation, or abundance. While many studies rely on traditional taxonomic identification methods, molecular-based methods are gaining favor representing rapid, less-subjective pathways for assessment of biodiversity. Total DNA from soil, water or invertebrate samples can be isolated and analysed using PCR with restriction enzyme digestion (PCR-RFLP), cloning and pyro-sequencing, or deep sequencing techniques, to establish DNA-based metrics that can then be linked to extant diversity. Whatever the chosen method, Bioline offers a suite of molecular tools to support the rapid characterisation of biodiversity across a range of environmental samples.
Population Growth and Climate Change– Managing the Future
One of the major concerns for agricultural and environmental scientist’s alike surrounds the management and support of the growing global population and the resulting impact on the environment and availability and health of environmental resources. Population has more than quadrupled over the past century and continues to rise, with a doubling in global food demand projected for the next 50 years. In addition to the basic challenges of increasing food production, it is the impact on the terrestrial and aquatic ecosystems that is critical to manage to ensure a sustainable future. For too long, humanity has demanded more than the regeneration capacity of the Earth’s biosphere and we are now witnessing the result of this overshoot – carbon-induced climate change, large-scale deforestation, collapsing fisheries, soil erosion and salinization, to name a few.
Environmental research efforts ultimately aim to integrate ecological studies with socioeconomic policies in order to affect the much needed changes in global and local policies. Meanwhile agricultural scientists are busy identifying ways to breed crops and livestock that are better placed to weather potential increases in temperature – introducing drought tolerance, salinity tolerance and other relevant characteristics to key crops and food supplies.
What is a stem cell?
Stem cells are undifferentiated cells, unique in both their proliferative capacity and ability to specialise into different cell types. The majority of cells that make up the human body have a specific function and lifespan and do not have the capacity to renew. In contrast, stem cells are able to divide and produce copies, replicating many times and serving as a biological repair system for the body, forming the basis of normal growth and development. In addition to their self-renewal properties, stem cells can differentiate to become more specific and specialized cell types. The classification of stem cells can be based upon their differentiating potential:
- Totipotent – capable of forming any human cell including placenta and giving rise to an entire functioning organism
- Pluripotent – capable of forming any human cell, except placenta, thus cannot give rise to an entire functioning organism
- Multipotent – capable of forming a limited range of human cells, usually within a specific tissue type
- Unipotent – capable of forming human cells along only one lineage
Stem cells can also be classified based on their origin and are present in both the embryo (embryonic stem cells) and adult (adult stem cells). Embryonic stem cells are derived from one of the earliest stages of development, thus they are pluripotent and can give rise to all human cell types. Adult stem cells are undifferentiated cells found within differentiated tissue, they are multipotent and can renew and become specialised to form any of the cell types of the tissue of origin. There are many different types of adult stem cell including:
- Mesenchymal – differentiate to form skin, muscle, bone, fat, cartilage, tendon and ligaments
- Hematopoietic – differentiate to form red and white blood cells as well as platelets
- Neural – differentiate to form cells within the nervous system
All adult stem cells are considered multi- or uni-potent, however recent advances in stem cell research have enabled adult stem cells to be re-programmed to become embryonic cell-like, expressing genes that define and maintain the same properties as embryonic cells. These induced pluripotent stem cells have been shown in research settings to be capable of generating cells characteristic of all three germ lines and are currently being used in drug development, disease modelling, and are hopeful candidates in the field of transplantation medicine.
Stem cell research areas
Stem cell researchers are interested in understanding the unique properties of stem cells, the drivers behind differentiation and self-renewal, as well as practical and therapeutic applications of stem cells in medicine. The range of cloning and molecular interrogation products offered by Bioline covers a full suite of tools to assist efforts towards better understanding of stem cells and their unique properties. Plus, ISO13485 manufacturing standards support seamless transition into therapeutic applications.
Understanding stem cell development and regulators of potency
What are the properties within a stem cell that allow for numerous cycles of self-renewal? How does a stem cell remain undifferentiated? What are the factors that induce a pluripotent cell towards differentiation into the many hundreds of different cell types within the human body? These are just some of the questions researchers are interested in understanding to shed light on the unique properties of stem cells, to understand how an embryo develops, or how hematopoietic stem cells balance self-renewal and the provision of differentiated progenitors for the maintenance of the blood system throughout life. Identifying the molecular cues that regulate stem cell systems, as well as the microenvironments that contribute the various triggers and maintenance systems, are key to gaining greater understanding of the unique properties of stem cells.
Induced pluripotent stem cells
Pluripotent stem cells offer limitless possibilities for understanding human development and tissue formation, as well as the opportunity to model diseases and understand disease mechanisms, with ultimate aims toward cell therapy and regenerative medicine. For many years embryonic stem (ES) cells were the sole source of pluripotent cells - carrying the weight and complication of associated ethical issues, the availability and utility of ES cells was ultimately limited. It was the Nobel-prize winning breakthrough from Takahashi and Yamanaka in 2006 that led to the identification of four important reprogramming factors that enabled generation of induced pluripotent stem cells (iPSCs) from previously differentiated somatic cells. From a simple skin biopsy or blood sample iPSCs can now be generated, offering genetically matched pluripotent cells for virtually any patient – not surprisingly, there is excitement around the potential of this field for understanding disease mechanisms and development of new treatment options. There are still many hurdles and details to understand and overcome, including the molecular characterisation of iPSCs and whether they are indeed equivalent to ES cells – evidence indicates differences in efficacy of differentiation as well as DNA methylation patterns – plus identifying effective protocols to achieve appropriate and reproducible differentiation is also proving to be a challenge.
Cancer stem cells
Through investigations into cancer metastases, disease recurrence, and acquired resistance to therapy, researchers have uncovered an enormous level of intratumoral heterogeneity, most likely due to a combination of genetic mutations and interactions with the tumour microenvironment, as well as the presence of what is now known as cancer stem cells (CSCs). Similar to normal stem cells, CSCs have self-renewal properties, however the strict regulations surrounding differentiation appear to be deregulated, resulting in continuous expansion and production of aberrant progeny – CSCs form tumours in healthy animals when transplanted, where normal stem cells do not. CSCs have now been identified in a broad range of blood and solid tissue tumours, including leukaemia, breast and brain cancers. While CSCs appear to represent a very small percentage of the tumour cell population, their existence has been used to explain how cancer cells are able to proliferate and acquire all the necessary mutations for cancer development, as well as the recurrent nature of many tumours, as CSCs appear to survive many cancer therapeutic efforts. Understanding CSCs and how they drive cancer development, as well as the mechanisms for how cancer patients acquire CSCs, form a major part of cancer research today.
Therapeutic usage of stem cells
Stem cells have been referred to as an “Aladdin’s Lamp” with rich promise to cure many diseases, however the current reality is far from this ideal. Efforts continue towards a better understanding of stem cell characteristics in order to harness this potential in the therapeutic realm. Today, stem cells are being used to produce differentiated cell lines for drug testing, previously only possible on animal models. Knowledge of the signalling mechanisms that control specific differentiation is still limited, and much more work can be done in this area to improve reproducibility and increase the range of available differentiated cell types. Harvesting of hematopoietic stem cells (HSCs) from bone marrow and more recently from circulating blood, has been used to treat blood cancer patients and replenish blood components after chemotherapy rounds.
Perhaps the most exciting potential for stem cell therapy lies in the generation of new cells and tissues to replace diseased or ailing counterparts. Known as cell-based therapy, this treatment is being looked at to regenerate spinal cord damage, brain injury and heart disease. With the advent of induced pluripotent stem cells, this approach to regenerative medicine is looking even more promising. From cloning and vector reagents, PCR and real-time PCR kits or individual components, sample preparation, sequencing and even whole assay design services, Bioline is ready with a full suite of tools to support the growing spectrum of stem cell research and application advances.
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.
Test a range of annealing temperatures. Depending on the qPCR results, the annealing temperature should be increased or decreased in 2-3oC increments. This can be done in a single experiment using a thermal gradient. Alternatively, a range of annealing temperatures should be tested using multiple qPCR experiments.
Thank you B.Dhungel, from Gallipoli Medical Research Foundation (GMRF), Greenslopes Private Hospital, University of Queensland for such a great review.
I am really impressed by the reproduciblity of the Bioline Isolate II RNA minikit, the cDNA sysnthesis and the Sybr-Lo Rox mix in qPCR. We consistently get high concentrations of pure RNA with 260/280 reading 2 when measured with the nanodrop.
We have seen that the cDNA synthesis kit from Bioline is able to synthesize high quality cDNA even from low starting RNA concentrations when analyzing the qPCR results.
Working with a new qPCR target
When looking at a new target of unknown expression level, the amount of RNA required to detect the target of interest depends on the abundance of the target in each sample. For high-copy-number transcripts you may be able to detect in as little as 10 pg, while for low-copy-number transcripts you may require more than 100 ng.
With all new targets (new sets of primers or new qPCR kits), a 10 fold serial dilution should always be run, to validate both the slope and the limit of detection (LOD) (the highest Ct value observed for a truly positive sample, as verified by melt-curve analysis). Only a single peak, which represents the specific PCR product, should be observed on the melt curve, the presence of other peaks indicates the presence of primer–dimers and/or nonspecific PCR products which can contribute to a stronger fluorescence signal and an earlier Ct, but will not be as sensitive.