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.
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!
MicroRNAs and Their Role in Personalised Medicine
Using mRNA qPCR panels as a tool to understand cancer
Circulating miRNAs are attracting interest in the burgeoning field of personalised medicine, with data supporting their diagnostic, prognostic and predictive biomarker potential. Effective miRNA profiling calls for reproducible, sensitive and specific tools with turn-around times fast enough to support investigations into what can be a rapidly changing disease progression and treatment environment. Introducing the latest miRNA RT-qPCR technology from Bioline, the EPIKTM Cancer miRNA Panel, offering sensitive SYBR® Green-based detection for 352 targets, including the most differentially expressed miRNAs and controls, for convenient, robust, and extremely specific miRNA analysis.
MicroRNAs in Cellular Processes and Disease
Since their discovery a little over 20 years ago1, miRNAs, previously overlooked within what was thought to be non-functional genome components, are now understood to be crucial regulators of important cellular functions2. The biogenesis of miRNA follows a complex path through a number of precursor forms resulting in the mature, single stranded miRNA which is about 20-25 nucleotides3. Mature miRNA interact with mRNA effecting post-translational gene regulation of cellular processes such as development, differentiation, proliferation, metabolism and apoptosis2, thus it is no surprise that aberrantly expressed miRNAs are a hallmark in many diseases, including cancer4. Recently, miRNAs were included within the traditional oncogene definition due to their vital role of controlling cell differentiation, proliferation and survival3, plus their role in the negative regulation of tumour suppressor genes is clearly evident4, and now over 12,600 publications are listed in the NCBI PubMed database relating miRNA with cancer5. Improvements in deep sequencing technology have allowed for genome-wide profiling of miRNA expression, revealing cancer-specific signatures that not only discriminate between cancer types with high accuracy, but identify tissues of origin in metastasised cancers4 – thus miRNA profiling as a cancer diagnostic became an attractive concept for development. Further, the highly regulated process of miRNA expression is sensitive to internal and external stimuli such as hormones, pharmacological molecules etc., leading to a unique miRNA profile within different tissue types, locations and time points, making them ideal candidates for prognostic and therapeutic oncology biomarkers5.
Liquid Biopsies and miRNA profiling
There is much excitement around the concept of ‘liquid biopsy’ – the ability to screen, monitor, and uniquely characterise tumours from, for example, a simple blood or plasma sample, foregoing the traditionally invasive, costly, and in many cases difficult to obtain tissue biopsy6. Although circulating cell-free tumour DNA (ctDNA) and circulating tumour cells (CTCs) are commonly the focus for these methods, circulating miRNAs are also attracting attention as viable candidates. Not only do they carry specific information about the patho-physiological state of an individual, miRNAs are remarkably stable in their protein-bound form and are present in cell-free body fluids such as plasma, serum, urine and saliva7. Correlations have been observed between specific circulating miRNAs and chemotherapy responses in a range of cancers5, and retrospective studies have also begun to identify miRNA signatures with strong predictive and prognostic potentials8. There are still many hurdles to overcome before miRNA profiling and liquid biopsy become mainstream diagnostic practice, not the least of which is establishing robust and reproducible protocols, in addition to identifying, among the growing list of candidates, which combination of targets can be linked to clinical relevance. Indeed, the very specificity with which cells express miRNA and the subsequent sensitivity to a range of stimuli, including age and gender, while attractive qualities for personalised medicine, make for a difficult moving target for applied research5.
Molecular Tools for miRNA profiling
Expression profile studies based on microarray platforms or large scale deep sequencing projects have been instrumental in the discovery and identification of miRNAs, serving to expand the current database to almost double the number of known human miRNAs in the past five years. Among the growing number of tools available for studying miRNAs, however, qPCR remains a routine favourite as it has potential to be extremely sensitive and accurate as well as being accessible to many in terms of access to instrumentation as well as overall cost. Notwithstanding the range of sample collection and extraction methods with all associated caveats, there are many challenges to overcome when applying RT-qPCR to miRNA. Targets are short, 18-22nt, as well as being highly homologous, often with as little as 1-2nt differences, in addition the mature miRNA target sequence is present in all the precursor forms9. EPIKTMmiRNA Panels are the latest release from Bioline that overcome these challenges and more, the unique assay design discriminates between even the most closely related miRNA targets, such as those within the let-7 family, with maximum sensitivity down to 10pg of total RNA, enabling low volume starting materials, such as required for blood and plasma samples and other liquid biopsy candidates10.
Advanced Design Concepts for miRNA qPCR Panels
There are many ways to overcome the difficulties of miRNA priming for RT-qPCR, many of which involve universal priming steps and additions of long tail nucleotides, resulting in a reduction of qPCR efficiency and target specificity9. EPIKTM miRNA Panel assays forego the universal priming approach and incorporate a unique 3-primer system, all of which are miRNA specific, including the initial RT stem-loop primer, allowing for clear discrimination of mature miRNAs as well as superior specificity to identify very closely related targets10. Despite the use of specific primer sets, the overall protocol is efficiently streamlined, less than 2 hours of instrument run-time leads to interpretation of profile changes in less than half a day, capitalising on the speed advantage that qPCR holds over sequencing or microarray approaches, and answering the turn-around time requirements of personalised medicine. To focus study efforts on the most differentially expressed miRNA candidates, panel design draws on a bioinformatics mega-study in collaboration with MiRXes, screening thousands of miRNA and cancer related articles and incorporating the top 340 targets. Additionally, assay performance is enhanced to ensure robust quantification over a 7-log linear dynamic range, covering low and high expressed targets within a single run10. In deference to the rising costs of laboratory technology, the RT step is linked to SYBR® Green detection, negating the use of sequence specific probes while maintaining specificity with the three specific primer approach.
As efforts continue towards the transition of miRNA profiling from bench to bedside, RT-qPCR panels offer an ideal tool to support current applied research and the resulting clinical processes. The latest technology in the field is represented by the strong assay design of the EPIKTM Cancer miRNA Panel, covering the most useful targets to monitor disease states, and offering the performance and sensitivity required for low level starting materials or liquid biopsy applications.
Bioline, The PCR Company, is proud to announce the worldwide release of the EPIK™ miRNA Panel Assays, developed for miRNA profiling of cancer samples, biofluids, and stem cells. The EPIK miRNA Panel Assays are the result of a collaboration between MiRXES in Singapore and Bioline.
Using a novel approach developed by MiRXES, combinations of miRNA specific reverse transcriptase and hemi-nested real-time PCR primers have been designed to maximize miRNA detection sensitivity and specificity. The real-time PCR assays use SensiSMART™ with SYBR® Green to enable detection of extremely low levels of miRNA with high specificity, allowing the discrimination between closely related miRNA sequences. This highly reproducible, sensitive platform has been validated independently by molecular diagnostic and pharmaceutical laboratories and showed superior performance to existing commercial assays. The EPIK miRNA Panel Assays provide researchers with a convenient way to quickly analyze miRNAs that suggest a correlation with the dysregulations in various cancers, in maintenance, differentiation and reprogramming of stem cells and to look at circulating miRNAs that are regarded as a promising new class of biomarkers.
Marco Calzavara, President of Bioline commented,
"The release of the EPIK miRNA Panel Assays follows the release of the ISOLATE II miRNA, Plant miRNA, Biofluids RNA, RNA/DNA/Protein and FFPE RNA/DNA Kits earlier this year and shows the continued commitment of Bioline to produce high quality miRNA kits to help researchers with miRNA expression profiling and quantification and in biomarker discovery."
EPIK™ miRNA Panel Assays confer high sensitivity, specificity and speed to the quantification of miRNAs and have been validated using both synthetic miRNA templates and human total RNA. Typically the assays detect as few as 100 copies of template per RT reaction with excellent assay efficiency and linearity to produce complete systems for miRNA profiling of cancer samples, biofluids and stem cells.
Lihan Zhou, CTO of MiRXES, commented,
“This exciting partnership combines our unique miRNA technology and Bioline’s vast experience in qPCR research tools to develop sensitive and robust assays to support the ever-growing community of miRNA researchers”.
MiRXES is a spin-off from the Bioprocessing Technology Institute of the Agency of Science Technology And Research (A*STAR) and the National University of Singapore. MiRXES collaborates extensively with academic, clinical and industrial partners in advancing the science and application of miRNA.
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:
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.