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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.
Epigenetics can be thought of in broad terms as the range of phenomena that bring phenotype into being without changing the underlying DNA sequence. Consider that the vast majority of cells within multicellular organisms have identical genotypes yet exhibit distinct cellular functions with different gene expression profiles that are stable and, in many instances, heritable. Thus the epigenetic landscape of an organism is an important function of cellular differentiation and much remains to be learned about these processes. Bioline offers a suite of molecular tools, proteomics reagents, assay panels, and complete assay design services to assist research efforts in the field of epigenetics.
Epigenetics – dynamic and global regulatory mechanisms
Our genome is not in a static state, and although our genotype strongly influences resulting phenotype, there are many differences in gene function and expression that cannot be attributed to changes or differences in DNA sequence alone. A range of epigenetic processes have been identified that post-synthetically modify DNA or interact with the proteins intimately associated with DNA, controlling the journey of transcription and ultimate translation of protein to subsequent phenotype. These mechanisms include a range of histone modifications that affect chromatin structure, subsequent DNA packing and chromosome formation, thus controlling the availability of genes to transcription mechanisms.
Methylation patterns of DNA have also been identified as strong drivers of gene activation/inactivation. Nearly all DNA methylation in mammalian genomes occur at cytosine residues of CpG dinucleotides, and high density CpG regions (or islands) that are methylated correlate with transcriptional repression. DNA methylation has been shown to play a role in important cellular processes including X chromosome inactivation, plus changes in methylated state have been identified in diseases such as cancer. DNA methylation patterns can change in response to environmental factors such as diet or exposure to toxins, and it has been shown that some of these adaptive changes are passed on to future generations. There are a range of methods to detect and quantify DNA methylation including PCR, Real-Time PCR, or High Resolution Melt (HRM) analysis, and Bioline provides reagents and kits to support research in these areas.
The role of RNA, in particular non-coding RNA (ncRNA), has been established as a major controlling element of multiple epigenetic events. In addition to the established functional RNAs (mRNA, tRNA, and rRNA) involved with processing of the coding RNA, and associated small nuclear and nucleolar (snRNA and snoRNA) RNAs that splice and modify RNA nucleotides, other small ncRNAs such as microRNAs (miRNA) and short interfering RNA (siRNA) have been identified in epigenetic mechanisms - involved in regulation of target mRNA and chromatin. Long non-coding RNAs have also been implicated in gene regulation, thus there is a complete network of ncRNAs intimately involved in epigenetic processes. These elements can be studied through deep genome sequencing approaches, as well as targeted PCR or Real-Time PCR assays to detect, identify, or quantify the various ncRNA elements, and Bioline offers a range of molecular tools for these analyses.
Epigenetics – the role of miRNAs
There are over 2,000 different short, single stranded RNA molecules currently identified as unique human mature miRNAs in the miRBase sequence database. After binding with the RNA-induced silencing complex (RISC), these miRNA complexes interact with target mRNA to induce degradation or cleavage, or to block translation – thus miRNAs are key epigenetic regulators of post-transcriptional gene expression. With involvement in many key cellular functions such differentiation, proliferation and apoptosis, the study of miRNAs and their mechanisms is key to understanding normal cellular development as well as aberrant disease states.
In addition to the full range of physiological and pathophysiological functions regulated by miRNA activity, they have also shown to be important regulators of other epigenetic mechanisms such as DNA methyltransferases and histone deacetylases. Conversely, DNA methylation and histone modification have been linked to regulation of expression of certain miRNAs, and there is also evidence of miRNAs regulating other miRNAs – thus an epigenetics-miRNA regulatory circuit exists, creating a complex and intricate gene expression process involving feedback mechanisms and elements of self-regulation. The regulatory landscape of miRNAs is far from straight forward and much remains to be understood regarding the expression of miRNAs and their subsequent regulatory roles. In addition to individual molecular tools and kits for isolation, amplification and identification of miRNA targets, Bioline in conjunction with MiRXES, a Singapore-based life science research company, have developed the EPIKTM range of miRNA assay panels representing key miRNA targets identified using extensive bioinformatics mega-studies and linked to important disease states such as cancer.
Epigenetics – Applications and Challenges
With epigenetic drivers involved in so many cellular processes, there are any number of applications in which to study the various mechanisms of DNA methylation, histone modification or non-coding RNA control of gene expression. Whether you’re sequencing an entire genome, isolating nucleic acids, amplifying target sequences or studying the resulting proteins, Bioline has the individual reagent, complete kit, individually designed assay service, or ready to use panel to support all of your research efforts.
Epigenetics and cell differentiation mechanisms
Understanding how a single cell grows into a complete multicellular organism still remains somewhat of a puzzle, how are the tissue specific patterns of gene expression established and maintained throughout the life of an organism? How do cells interpret signalling cues, and how do stem cells both maintain their stem cell properties and differentiate progeny into various cell lineages? Given that the majority of cells within this system contain genetically identical material it is clear that epigenetic factors play a key role during organism development, as well as in stem cell renewal and differentiation throughout life. There is still much to be understood about the drivers behind embryogenesis, as well as the signals that fine-tune cell functions and gene expression changes throughout the lifespan of an organism. Although key genes and regulators of those genes have been identified in these processes, any number of epigenetic factors are involved, simultaneously interacting with each other and the local and external environment to bring about the expression of the unique set of genes that define each and every one of us.
Understanding epigenetic inheritance
Many epigenetic changes within cells act as one-way barriers, ensuring that cell-type specification and activity is stable and maintained, thus there are clear examples for mitotic inheritance of epigenetic regulation. Mechanisms exist for propagation of DNA methylation patterns, however little is known about how epigenetic flexibility is maintained during cell development and differentiation, and non-DNA based epigenetic inheritance is still poorly understood. Meiotic inheritance is even more controversial. During mammalian sexual reproduction, the epigenome must be reset back to a totipotent state in preparation for the development of the next generation. Given this erasure of somatic epigenetic signatures, it is difficult to understand how epigenetic changes could be transgenerationally, or meiotically inheritable. Intriguing multi-generational studies have reportedly identified specific epigenetic markers and associated phenotypic characteristics in offspring linked to environmental exposures such as diet restriction or toxin exposure in grand-parents, suggesting transgenerational epigenetic effects may indeed exist. However many studies in this area have been contested, and a mechanism for such inheritance has not yet been defined. There is certainly a wide gap in our knowledge and research efforts persist with mixture of excitement and caution.
Epigenetics in cancer and disease states
Given their key role in so many important cellular functions it stands to reason that aberrant epigenetic processes have been identified in many cancers as well as other disease states. Hyper-methylation of CpG islands has been identified in a range of cancers resulting in subsequent silencing of tumour suppressor genes, DNA repair genes or other processes important to normal cell growth. Epigenetic factors have also been linked to inherited disorders associated with mental retardation such as Fragile X syndrome, Prader-Willi, and Rett syndrome. Epigenetic changes have been observed in response to external environmental factors, and as such, epigenetic links are being investigated for conditions such as obesity, diabetes or heart disease. Although there are many clear correlations between epigenetic states and disease, there are many more confounding studies and results that have been difficult to replicate. There is vast variation in epigenetic alterations across cell populations within an individual, let alone across populations, meaning that many epigenetic studies of disease bring up more questions than they answer. In addition, it is often difficult to determine whether epigenetic changes are a cause or consequence of disease state. Nevertheless, epigenetic profiling continues, and epigenetic therapeutics are also being developed, as efforts continue towards a greater understanding of these important regulatory elements and their roles in disease.
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.
Simon Baker PhD, Global R&D Director, Bioline Reagents Ltd: discusses MicroRNA Profiling Using a Rapid and Highly Sensitive qPCR Panel.
If you missed the live Webinar, you can view it here or follow the link below to download it and view it at your leisure.
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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.
It’s that time of year again!!!! We are proud to announce Bioline is supporting iGEM teams in 2015. Our already competitive pricing drops 40% for the young and enthusiastic iGEM researchers in the United Kingdom.
‘Sponsorship is very difficult, particularly for small projects such as ours. Bioline are very kindly sponsoring us. A big thank you to them.’ - York iGEM 2014 Team
Popular tools used by successful iGEM teams around the world include:
- Bioline Competent Cells and Cloning Reagents - Transform plasmids from the iGEM kit plates with extremely efficient alpha-select silver or gold efficiency cells.
- MyTaq™ HS - Use MyTaq HS to amplify DNA from your minipreps in as little as 30 minutes, or amplify directly from your colonies! See the MyTaq colony PCR application note or this SOP written by the Danish iGEM2013 team.
- ISOLATE II Plasmid Mini Kit - Pure and very high DNA yields from your transformed colonies.
- VELOCITY DNA Polymerase - Perfect for all your high-fidelity applications.
- HyperLadder™ DNA Markers - Very popular Bioline range of markers for DNA sizing, as used by Craig Venter's research team.
- Real-time PCR Kits - Trusted SensiFAST™ kits for gene expression analysis.
- Core Reagents - High quality buffers, agarose and other essentials.
Get in touch with your local UK Bioline Account Manager to enquire about an iGEM offer pricing plan.
Then, why not let your fellow iGEM researchers know about this great offer using the sharing links below? Also feel free to Tweet or email us if you have any further questions www.bioline.com/social We look forward to hearing from you soon and we wish all iGEM2015 entrants every success in this year's competition!
Terms and Conditions
This offer is valid on any product in our range for any iGEM team in the United Kingdom. No minimum or maximum spend to qualify for the discount. Expires on September 30, 2015. Not valid with any other promo codes or special offers. Please contact us for details of discounts available to iGEM teams in the United States, Germany, France, Singapore and Australia.