Francine Z MarquesBaker Heart and Diabetes Institute · Heart Failure Research Group
Francine Z Marques
BSc (Hons), M Mol Bio, PhD
About
200
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Introduction
Additional affiliations
April 2017 - April 2021
September 2007 - October 2011
Publications
Publications (200)
The gut‐immune axis is a relatively novel phenomenon that provides mechanistic links between the gut microbiome and the immune system. A growing body of evidence supports it is key in how the gut microbiome contributes to several diseases, including hypertension and cardiovascular diseases (CVDs). Evidence over the past decade supports a causal lin...
Purpose of the Review
To review what intestinal permeability is and how it is measured, and to summarise the current evidence linking altered intestinal permeability with the development of hypertension.
Recent Findings
Increased gastrointestinal permeability, directly measured in vivo, has been demonstrated in experimental and genetic animal mode...
The gut microbiome regulates many important host physiological processes associated with cardiovascular health and disease; however, the impact of the gut microbiome on aldosterone is unclear. Investigating whether gut microbiota regulate aldosterone can offer novel insights into how the microbiome affects blood pressure. In this study, we aimed to...
According to several international, regional, and national guidelines on hypertension, lifestyle interventions are the first-line treatment to lower blood pressure (BP). Although diet is one of the major lifestyle modifications described in hypertension guidelines, dietary fiber is not specified. Suboptimal intake of foods high in fiber, such as in...
Introduction
Animal models are regularly used to test the role of the gut microbiome in hypertension. Small-scale pre-clinical studies have investigated changes to the gut microbiome in the angiotensin II hypertensive model. However, the gut microbiome is influenced by internal and external experimental factors which are not regularly considered in...
Highlights
•HFpEF is a complex clinical syndrome with incompletely understood pathophysiology. The recent demonstration of a beneficial impact of sodium glucose co-transporter 2 inhibitor therapy in HFpEF has drawn attention to the potential role of deranged cardiac metabolism, although this remains poorly understood.
•Arterial and coronary sinus b...
Background: Traditional cardiovascular risk factors, including hypertension, only explain part of major adverse cardiac events (MACE). Understanding what other risk factors contribute to MACE is essential for prevention. Constipation shares common risk factors with hypertension and is associated with an increased risk of several cardiovascular dise...
Hypertension, defined as persistently elevated systolic blood pressure (SBP) >140 mmHg and/or diastolic blood pressure (DBP) at least 90 mmHg (International Society of Hypertension guidelines), affects over 1.5 billion people worldwide. Hypertension is associated with increased risk of cardiovascular disease (CVD) events (e.g. coronary heart diseas...
Background:
Gastroparesis (GP) is characterized by delayed gastric emptying in the absence of mechanical obstruction.
Objective:
Genetic predisposition may play a role; however, investigation at the genome-wide level has not been performed.
Methods:
We carried out a genome-wide association study (GWAS) meta-analysis on (i) 478 GP patients from...
Alzheimer’s disease (AD) is a neurodegenerative disorder with poorly understood aetiology. AD has several similarities with other ‘Western lifestyle’ inflammatory diseases, where the gut microbiome and immune pathways have been associated. Previously, we and others have noted the involvement of metabolite-sensing G protein coupled receptors (GPCRs)...
Gut microbiome research has increased dramatically in the last decade, including in renal health and disease. The field is moving from experiments showing mere association to causation using both forward and reverse microbiome approaches, leveraging tools such as germ-free animals, treatment with antibiotics, and faecal microbiota transplantations....
Dietary fibre intake is associated with fewer cases of ischaemic stroke. This is likely via the microbiota-gut-brain axis, where fibre is fermented by the gut microbiota, releasing short-chain fatty acids (SCFAs). However, whether fibre or SCFAs can reverse adverse post-stroke outcomes remains unknown. Here, we demonstrated that a low fibre diet ex...
Introduction
Gut microbiome studies often overlook experimental factors that could influence gut microbiome diversity and could impact findings. Large-scale studies investigating these experimental factors are lacking. Thus, we aimed to determine which experimental factors influence the gut microbiome diversity in pre-clinical animal model studies....
We provide evidence that stroke-induced gut breakdown results in bacteria translocation to the ischaemic mouse brain. Inhibition of sympathetic tone reduced bacterial load in the post-stroke brain and reduced functional deficits without altering cerebral apoptosis, neuroinflammation or infarct volume. These findings indicate that the activation of...
Fermentation of dietary fibre by the gut microbiota leads to the production of metabolites called short-chain fatty acids (SCFAs), which have emerged as potent regulators of immune, metabolic, and tissue barrier functions. More recently, a high fibre diet and SCFA supplementation were shown to lower blood pressure and be cardio-protective. SCFAs ac...
Diet and the gut microbiota have a profound influence on physiology and health, however, mechanisms are still emerging. Here we outline several pathways that gut microbiota products, particularly short-chain fatty acids (SCFAs), use to maintain gut and immune homeostasis. Dietary fibre is fermented by the gut microbiota in the colon, and large quan...
Background:
Raised blood pressure (BP) remains the single most important modifiable risk factor contributing to cardiovascular and all-cause mortality in Australia and worldwide. May Measurement Month, a global BP measurement and screening campaign initiated by the International Society of Hypertension and carried out in Australia since its incept...
A large body of evidence has emerged in the past decade supporting a role for the gut microbiome in the regulation of blood pressure. The field has moved from association to causation in the last 5 years, with studies that have used germ-free animals, antibiotic treatments and direct supplementation with microbial metabolites. The gut microbiome ca...
Fibers remain undigested until they reach the colon, where some are fermented by gut microbiota, producing metabolites called short-chain fatty acids (SCFAs), such as acetate and butyrate1. SCFAs lower blood pressure in experimental models2–5, but their translational potential is unknown. Here we present the results of a phase II, randomized, place...
Dietary fibre regulates blood pressure (BP) through gut microbial production of acidic metabolites called short-chain fatty acids (SCFAs). The acidic environment in the large intestine activates a proton-sensing G-protein coupled receptor, GPR65. We aimed to characterize the luminal pH in the intestine of hypertensive patients and normotensive indi...
In the last 7 years, we have seen a large body of evidence showing that the gut microbiota, the microorganisms that inhabit the intestine, have a role in experimental and clinical blood pressure (BP) regulation. Robust evidence comes from forward and reverse microbiome experiments, where faecal microbiota transplantation and treatment with specific...
The gut microbiome has been recently discovered to play a role in blood pressure (BP) regulation. Short-chain fatty acids (SCFAs), produced from the fermentation of fibre by the gut microbiota, are protective in experimental hypertension models. However, whether the protective effects of fibre are dependent on SCFA-receptor (GPR41, GPR43) signallin...
Blood pressure(BP) management interventions have been shown to be more effective when accompanied by appropriate patient education. As high BP remains poorly controlled, there may be gaps in patient knowledge and education. Therefore, this study aimed to identify specific content and delivery preferences for information to support BP management amo...
Clinical evidence supports that dietary fibre intake lowers blood pressure (BP). Dietary fibres remain intact until they reach the colon. There, they are fermented by gut microbial communities to produce metabolites, particularly short-chain fatty acids (SCFAs). Previous studies, including our own, have demonstrated that supplementation with the SC...
Blood pressure (BP) variability is an independent risk factor for cardiovascular events. Recent evidence supports a role for the gut microbiota in BP regulation. However, whether the gut microbiome is associated with BP variability is yet to be determined. Here, we aimed to investigate the interplay between the gut microbiome and their metabolites...
Low fibre intake has emerged as an important risk factor for hypertension through changes in the gut microbiota, but the specific metabolites involved in this phenotype are unknown. We identified two novel secondary metabolites, p-Cresol glucuronide (PCG) and p-Cresol sulfate (PCS) that were elevated in plasma samples in mice on a low fibre diet. T...
Altered immune cell activation plays a key role in promoting hypertension. A major question remains as to the immune system becomes activated during hypertension. A possibility is necroptosis, which a newly described form of cell death alerts the immune system to dying cells, causing inflammation. Using mice genetically unable to undergo necroptosi...
Dietary fibre intake is negatively associated with stroke and overall mortality. A plausible hypothesis is that this happens via the gut microbiota, producing metabolites known as short-chain fatty acids (SCFAs) during fibre fermentation. Indeed, our unpublished data showed that low-fibre (LF) intake worsens stroke outcomes while high-fibre (HF) in...
Dietary fibre regulates blood pressure (BP) through gut microbial production of acidic metabolites known as short-chain fatty acids (SCFAs). The specific mechanisms of how SCFAs regulate BP are still emerging. We hypothesised that acidic metabolites that are abundant in the large intestine may activate proton-sensing G-protein coupled receptors, su...
Objective: Altered immune cell activation plays a key role in promoting hypertension. How the immune system becomes activated during hypertension is unknown. A possibility is necroptosis, a newly described form of cell death that alerts the immune system to dying cells, causing inflammation. Using Mlkl -/- mice that are genetically unable to underg...
Blood pressure(BP) management interventions have been shown to be more effective when accompanied by appropriate patient education. As high BP remains poorly controlled, there may be gaps in patient knowledge and education. Therefore, this study aimed to identify specific content and delivery preferences for information to support BP management amo...
Purpose of Review
To discuss the interplay behind how a high-fibre diet leads to lower blood pressure (BP) via the gut microbiome.
Recent Findings
Compelling evidence from meta-analyses support dietary fibre prevents the development of cardiovascular disease and reduces BP. This relation is due to gut microbial metabolites, called short-chain fatt...
In May 2022, Nature Reviews Cardiology published our comprehensive roadmap of strategies to support the career progression of cardiovascular researchers,¹ driven by the Australian Cardiovascular Alliance Emerging Leaders Committee. The path to this roadmap started in 2019, when we discovered only 23% of Australian cardiovascular researchers had per...
Background:
Blood pressure (BP) variability is an independent risk factor for cardiovascular events. Recent evidence supports a role for the gut microbiota in BP regulation. However, whether the gut microbiome is associated with BP variability is yet to be determined. Here, we aimed to investigate the interplay between the gut microbiome and their...
Atopic dermatitis (AD) is a globally prevalent skin inflammation with a particular impact on children. Current therapies for AD are challenged by the limited armamentarium and the high heterogeneity of the disease. A novel promising therapeutic target for AD is the microbiota. Numerous studies have highlighted the involvement of the skin and gut mi...
After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with develop...
Cardiovascular disease remains the leading cause of death worldwide. Cardiovascular research has therefore never been more crucial. Cardiovascular researchers must be provided with a research environment that enables them to perform at their highest level, maximizing their opportunities to work effectively with key stakeholders to address this glob...
High-heat processed foods contain proteins that are partially resistant to enzymatic digestion and pass through to the colon. The fermentation of resistant proteins by gut microbes produces products that may contribute to chronic disease risk. This pilot study examined the effects of a resistant protein diet on growth, fecal microbiome, protein fer...
Blood pressure (BP) variability is an independent risk factor for cardiovascular events. Recent evidence supports a role for the gut microbiota in BP regulation. However, whether the gut microbiome is associated with BP variability is yet to be determined. Here, we aimed to investigate the interplay between the gut microbiome and their metabolites...
Cardiovascular disease is one of the most significant causes of death globally, especially in regions where unhealthy diets are prevalent and dietary fibre intake is low.1,2 Fibre, particularly prebiotic types that feed gut microbes, is essential for maintaining healthy gut microbial ecosystems.3 One assumption has been that cardiovascular health r...
Besides damaging the brain, stroke causes systemic changes, including to the gastrointestinal system. A growing body of evidence supports the role of the gut and its microbiota in stroke, stroke prognosis, and recovery. The gut microbiota can increase the risk of a cerebrovascular event, playing a role in the onset of stroke. Conversely, stroke can...
Background: Recent evidence indicates that high numbers of cardiovascular (CV) researchers have considered leaving the research and academic sector due to lack of job security and low funding success. Thus, there is an urgent need to develop solutions to support the retention of early- and mid-career researchers (EMCRs). Here, we aimed to explore t...
We have shown that systemic and cardiac sympathetic activation is present in heart failure with preserved ejection fraction (HFpEF) patients. Conversely, whereas systemic inflammatory activation was also detected in HFpEF, we did not detect local myocardial release of inflammatory cytokines. Activation of the sympathetic system correlated with both...
MicroRNA miR-181a is downregulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here, we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecul...
Elevated blood pressure (BP), or hypertension, is the main risk factor for cardiovascular disease. As a multifactorial and systemic disease that involves multiple organs and systems, hypertension remains a challenging disease to study. Models of hypertension are invaluable to support the discovery of the specific genetic, cellular and molecular mec...
High fibre (HF) diet protects against hypertension via the production of acidic metabolites, e.g. short-chain fatty acids, by the gut microbiota. While these metabolites have a direct role in blood pressure (BP) regulation, their acidic nature may activate proton-sensing receptors, which have anti-inflammatory functions. G-protein coupled receptor...
Dietary fibre is fermented by the gut microbiota and protects against the development of cardiovascular disease (CVD) through the production of gut microbial metabolites. We hypothesised dietary fibre intake during pregnancy may prevent the development of CVD in the offspring via in utero epigenetic mechanisms. To investigate this, we fed C57BL/6J...
High dietary fibre is fermented by the gut microbiota, resulting in the release of metabolites called short-chain fatty acids (SCFAs). Both fibre and SCFAs can reduce high blood pressure (BP) and its associated cardio-renal complications. However, the underlying mechanisms remain unclear. SCFAs can be detected by metabolite-sensing receptors GPR41...
Recent findings in experimental models have shown that the microRNA miR-132 (mir-132) is an important regulator of liver homeostasis and lipid metabolism. We aimed to assess miR-132 expression in liver and fat tissues of obese individuals and examine its association with blood pressure (BP) and hepatic steatosis. We examined obese individuals under...
Recent evidence supports a role for the gut microbiota in hypertension, but whether ambulatory blood pressure is associated with gut microbiota and their metabolites remains unclear. We characterized the function of the gut microbiota, their metabolites and receptors in untreated human hypertensive participants in Australian metropolitan and region...
Aim
Gut microbiota-derived metabolites, such as short-chain fatty acids (SCFAs) have vasodilator properties in animal and human ex vivo arteries. However, the role of the gut microbiota and SCFAs in arterial stiffness in humans is still unclear. Here we aimed to determine associations between the gut microbiome, SCFA and their G-protein coupled sen...
Background
Hypertension is a prevalent chronic disease worldwide that remains poorly controlled. Recent studies support the concept that the gut microbiota is involved in the development of hypertension and that dietary fibre intake may act through the gut microbiota to lower blood pressure (BP). Resistant starch is a type of prebiotic fibre which...
Aims: The current study investigates the role of diet in mediating the gut microbiome-cardiovascular association which has not yet been explored in humans.
Methods and Results: Using a two-arm dietary intervention study in healthy participants ( N = 70), we assessed the effects of omega-3 and fibre supplementation on gut microbiome composition and...
Background
Risk factors for heart failure with preserved ejection fraction (HFpEF) include hypertension, age, sex, and obesity. Emerging evidence suggests that the gut microbiota independently contributes to each one of these risk factors, potentially mediated via gut microbial‐derived metabolites such as short‐chain fatty acids. In this study, we...
pH sensing by GPR65 regulates various inflammatory conditions, but its role in skin remains unknown. In this study, we performed a phenome-wide association study and report that the T allele of GPR65-intronic single-nucleotide polymorphism rs8005161, which reduces GPR65 signaling, showed a significant association with atopic dermatitis, in addition...
Maternal high-fat diet in rabbits leads to hypertension and elevated renal sympathetic nerve activity (RSNA) in adult offspring but whether this is due to adiposity or maternal programming is unclear. We gave intracerebroventricular (ICV) and ventromedial hypothalamus (VMH) administration of leptin-receptor antagonist, α-melanocyte-stimulating horm...
May Measurement Month (MMM) is an annual global blood pressure (BP) screening campaign aimed at obtaining standardized BP measurements and other relevant health information from members of the community to increase awareness of elevated BP and the associated risks. Adults (≥18 years) were recruited through opportunistic sampling across the various...
Objective
High blood pressure (HBP) is a major risk factor for cardiovascular disease (CVD) and the prevalence of both is higher in regional versus metropolitan areas. The gut microbiome has emerged as important contributor to HBP, however, the relationship between BP and specific gut microbes and their metabolites is unclear. Here we aimed to dete...
Objective
Fibre intake is associated with lower incidence of hypertension and cardiovascular mortality. Prebiotic fibre, such as resistant starches and soluble fibre, are fermented by commensal gut microbiota to short-chain fatty acids (SCFAs), which have a protective role in cardiovascular disease. We, and now others, have shown that supplementati...
Objective
Dietary fibre lowers blood pressure (BP) and risk of cardiovascular disease and death via production of acidic metabolites by the gut microbiota. The mechanisms involved, however, are still elusive. Here, we aimed to understand the role of intestinal pH and the proton-sensing receptor GPR65 in the cardiovascular protection by dietary fibr...
Objective
Cardiovascular disease is the leading cause of death in Australia, yet funding has stagnated in recent years, resulting in economic challenges within the cardiovascular research sector. Here we investigated the challenges faced by cardiovascular researchers in Australia as an initiative of the Emerging Leaders Committee of the Australian...
Objective
Ganaxolone is a synthetic form of allopregnanolone, an allosteric modulator of gamma aminobutyric acid type A receptors (GABAAR). Allopregnanolone reduced blood pressure (BP) in male BPH/2J mice with neurogenic hypertension (Stevenson et al., 2017), which suggests that a lack of tonic inhibition in the forebrain mediates the hypertension....
MicroRNA miR-181a is down-regulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecul...
Aims: Recent evidence supports a role for the gut microbiota in hypertension, but whether ambulatory blood pressure (BP) is associated with gut microbiota and their metabolites remains unclear. Here we characterised the function of the gut microbiota, their metabolites and receptors in untreated human hypertensive participants in metropolitan and r...
Amongst other immune cells, neutrophils play a key role in systemic inflammation leading to cardiovascular disease and can release inflammatory factors, including lipocalin-2 (LCN2). LCN2 drives cardiac hypertrophy and plays a role in maladaptive remodelling of the heart and has been associated with renal injury. While lifestyle factors such as die...
Background: Hypertension is a prevalent chronic disease worldwide that remains poorly controlled. Recent studies support the concept that the gut microbiota is involved in the development of hypertension, and that dietary fibre intake may act through the gut microbiota to lower blood pressure (BP). Resistant starch is a type of prebiotic fibre whic...
There is increasing evidence of the influence of the gut microbiota on hypertension and its complications, such as chronic kidney disease, stroke, heart failure, and myocardial infarction. This is not surprising considering that the most common risk factors for hypertension, such as age, sex, medication, and diet, can also impact the gut microbiota...
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Increasing evidence supports a role for the gut microbiota in the development of cardiovascular diseases such as hypertension and its progression to heart failure (HF). Dietary fibre has emerged as a modulator of the gut microbiota, resulting in the release of gut metabolites called short-chain fatty acids (SCFAs), such as acetate. We have shown pr...
Low fermentable fibre intake has emerged as an important risk factor for hypertension through changes in the gut microbiota, but the biological pathways and specific metabolites involved are unknown. We performed untargeted liquid-chromatography mass spectrometry metabolomic profiling on plasma samples from 16 mice fed low or high fibre diets and 7...
Background
Cardiovascular disease is the leading cause of death in Australia. Investment in research solutions has been demonstrated to yield health and a 9.8-fold return economic benefit. The sector, however, is severely challenged with success rates of traditional peer-reviewed funding in decline. Here, we aimed to understand the perceived challe...
Purpose of review:
To summarize the recent evidence that supports a role for the gut microbiota, microbiota-derived metabolites, and dysbiosis on cardiovascular risk factors, and to discuss the neuro-cardio-metabolic mechanisms that link gut microbiota and heart failure.
Recent findings:
There is growing evidence that the gut microbiota communic...
Ganaxolone is a synthetic form of allopregnanolone, an allosteric modulator of gamma aminobutyric acid type A receptors (GABA A R). Allopregnanolone reduced blood pressure (BP) in male BPH/2J mice with neurogenic hypertension (Stevenson et al., 2017), which suggests that a lack of tonic inhibition in the forebrain mediates the hypertension. The hyp...
Background: High blood pressure (BP) continues to be a major, poorly controlled but modifiable risk factor for cardiovascular death. Amongst key Western lifestyle factors, a diet poor in fibre is associated with prevalence of high BP. The impact of lack of prebiotic fibre and the associated mechanisms that lead to higher BP are unknown. Here we sho...
: The New Investigators Committee (NIC) of the International Society of Hypertension (ISH) is a dynamic group of junior doctors and scientists, actively involved in various society activities. This report highlights the events (scientific meetings and summer schools) and activities (social media, mentorship and networking) during 2019 including May...
Abstract There has been intense interest in the role of the gut microbiome in human health and a broad range of diseases in recent years. In the context of cardiovascular disease, gut dysbiosis (defined as a change in the gut microbiome and the gut-epithelial barrier) has been linked to disturbances in blood pressure (BP) regulation. These findings...
It has been 45 years since Gunther Schlager used a cross breeding program in mice to develop inbred strains with high, normal, and low blood pressure (BPH/2, BPN/3, and BPL/1 respectively). Thus, it is timely to gather together the studies that have characterized and explored the mechanisms associated with the hypertension to take stock of exactly...
Over the past decade, the immune system has emerged as an important component in the aetiology of hypertension. There has been a blooming interest in the contribution of the gut microbiota, the microbes that inhabit our small and large intestine, to blood pressure (BP) regulation. The gastrointestinal tract houses the largest number of immune cells...
Schlager mice (BPH/2J) are hypertensive due to a greater contribution of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS). The kidneys of BPH/2J are hyper-innervated suggesting renal nerves may contribute to the hypertension. We therefore determined the effect of bilateral renal denervation (RD) on hypertension in BPH/2J. Mea...
Purpose of review:
To summarize evidence supporting that microorganisms colonizing our gastrointestinal tract, collectively known as the gut microbiota, are implicated in the development and maintenance of hypertension in experimental models.
Recent findings:
The use of gnotobiotic (germ-free) mice has been essential for advancement in this area...
Background and aims:
Fetal and postnatal growth restriction predispose to cardiovascular disease (CVD) in adulthood. Telomeres are repetitive DNA-protein structures that protect chromosome ends, and the loss of these repeats (a reduction in telomere length) is associated with CVD. As exercise preserves telomere length and cardiovascular health, th...
Study question:
Are any microRNAs (miRNAs) that target the placental renin-angiotensin system (RAS) in the human placenta suppressed in early gestation?
Summary answer:
Overall, 21 miRNAs with predicted RAS mRNA targets were less abundant in early versus term placentae and nine were more highly expressed.
What is known already:
Regulation of h...
While aging is a critical risk factor for heart failure, it remains uncertain whether the aging heart responds differentially to a hypertensive stimuli. Here we investigated phenotypic and transcriptomic differences between the young and aging heart using a mineralocorticoid-excess model of hypertension. Ten-week (“young”) and 36-week (“aging”) mic...
Hypertrophic cardiomyopathy thickens heart muscles reducing functionality and increasing risk of cardiac disease and morbidity. Genetic factors are involved, but their contribution is poorly understood. We used the hypertrophic heart rat (HHR), a unique normotensive polygenic model of cardiac hypertrophy and heart failure to investigate the role of...
Objectives:
Noradrenaline released from sympathetic nerves is rapidly inactivated via the action of the noradrenaline transporter (NET). We aimed to determine whether a single nucleotide polymorphism (SNP) in the NET gene, rs7194256, was associated with blood pressure and plasma noradrenaline concentration in patients with resistant hypertension....
Questions
Questions (4)
I am trying to PCR amplify the complete mRNA (from first ATG to 3'UTR) from a human gene using genomic DNA. The total fragment has 3296 bp. I need the fragment to be until the end of the 3'UTR as this is where a SNP of interest is. My intention is to clone this fragment into a plasmid to do functional experiments. The fragment has 53.4% GC. I have tried to order the primers twice now in case there was something wrong with them.
I have tried the following enzymes:
- Bioline Immolase
- Bioline MyTaqHS
- KAPA2G Robust PCR kit and KAPA2G Robust HotStart (with both buffers A and B, enhancer...)
All these enzymes should be fine for fragments <5kb.
I either get no bands with most enzymes or with KAPA enzymes I get several non-specific bands, the largest being about 2 kb (so no bands of correct size).
I have attached the primers and sequence. If anyone could help me it would be highly appreciated, I have ran out of solutions.
Best wishes,
Francine
Dear all,
We experience regular issues in our lab because one member is very scared of ethidium bromide (even thought we have shown by calculations that the concentration used does not pose a health risk). We run our gels (stained with either ethidium bromide at low concentration or SYBR safe) in 1xTAE buffer (which does not contain any added ethidium bromide or SYBR safe).
1- Would this buffer pose a health risk after it has in contact with the gel?
2- What is the best way to dispose the TAE buffer after running the gel?
If you could provide me with references that would be great.
Thanks a lot for your help,
Francine
I am interested in a particular microRNA produced in the kidney but also found in the circulation. I would like to isolate circulating exosomes and then identify by surface markers those excreted by the kidney. Does anyone know if there is such a marker? I haven't been able to find it. Thanks heaps in advance.
I have found a few mutations in the upstream region of my gene of interest, and I would like to test in vitro whether they have a role in the regulation of the expression of this gene. I have done heaps of cloning, in vitro work, luciferase assays, etc for miRNA work but I am not so experienced with upstream regulatory regions. I would appreciate some help with the following:
-what plasmid would be the best to use?
- with the cloning, do I clone the whole region, or a small region containing each mutation?
- I assume I would need to make a plasmid containing the same region but without the mutations to use as controls?
- Do I need to co-transfect the cells with anything (for example, a transcription factor predicted to bind to this region)?
