Interview conducted by April Cashin-Garbutt, BA Hons (Cantab)
Please can you give a brief introduction to miRNA and RNA interference?
RNA interference is a process in which RNA molecules inhibit gene expression. This gene silencing process is controlled by RNA-induced silencing complex or RISC and is initiated by small duplex RNA molecules or miRNA that is present inside cell plasma.
These RNA molecules may come from outside of the cell from viruses, for example, or they may have been transfected into cells for research. They can also be encoded by the cell’s own genome.
This double-stranded RNA is cleaved into shorter fragments by Dicer enzymes. It is then separated into single strands, which are incorporated into an active RISC complex. This complex targets the mRNA, which is then cleaved and therefore cannot be used as a template for protein synthesis. Consequently there is a down-regulation of gene expression.
miRNAs are short non-coding RNAs that are present in the genome and they regulate gene expression, especially during development. They are very conserved in eukaryotic organisms, and that’s thought to be a vital and evolutionary ancient component of genetic regulation.
What percentage of mammalian gene expression is thought to be regulated by miRNA?
It is thought that the human genome includes over one thousand different miRNAs and that around 60% of human genes are regulated by miRNAs. These miRNAs are abundant in many cell types.
Why do miRNAs offer potential as biomarkers?
Research has shown that miRNAs play an important role in various cellular processes such as apoptosis, differentiation and development.
Aberrant expression of miRNA has also been implicated in numerous disease states and recent studies have shown miRNA is misexpressed in human cancers, where they might act as an oncogene or a tumor suppressor.
miRNAs have also been implicated in heart disease, obesity. They also appear to be involved in the control of the nervous system.
In the last few years, miRNA expression profiling studies, using microarrays, have been performed that can differentiate normal tissue from cancer tissue through a unique miRNA expression signature. These differences can be used to clarify different tumor types and tumor grades.
Certain miRNA signatures have even been correlated with prognosis and could potentially be used to determine the specific course of treatment. There are even miRNA based therapies under investigation.
Together, these make miRNAs potential biomarkers.
Please can you outline the range of products AMSBIO have developed for stable expression of mature miRNAs?
We have human and mouse miRNA lentivirus and the miRNAs we offer are the ones that are listed in miRBase database. What we have done is amplified the miRNA precursors and their native context sequences both upstream and downstream from the miRNA and cloned them into a lentivector.
In this lentivector the expression is controlled by optional inducible CMV promoter, the co-expressing GFP and pre-miRNA are separated by sequence and in this case, the GFP provides a convenient indicator of miRNA expression levels. There is also a puromycin antibiotic selection marker in the vector which is good for stable expression. We also provide negative control of lentivirus.
How do miRNA inhibitors down-regulate miRNA activity?
They work primarily through a blocking mechanism. The anti-miRNA oligonucleotides that we have bind to the miRNA (they are synthetic reversed complements) and prevent the miRNA being used for the RNA interference process.
What applications can anti-miRNA be used for?
They can be used for miRNA functional analysis by down-regulating miRNA activity. This includes the analysis of miRNA target sites, identification and validation, screening for miRNAs that regulate gene expression and screening for miRNAs that affect cellular processes.
Could you please outline AMSBIO’s range of anti-miRNA lentiviruses and how they were developed?
We offer anti-micro RNA lentiviruses for all miRNAs listed in the miRBase database . They are expressed under optional inducible H1 promoter or constitutive U6 promoter. We also offer several markers, including fluorescent ones, and negative control lentivirus.
What are AMSBIO’s plans for the future?
We plan to expand our portfolio of these lentivirus products. We also offer a wide range of lentiviral particles that express human and mouse cDNAs as well as fluorescent lentiviral particles and ones that express shRNA.
Another focus of our company is physiologically relevant cell culture and we offer different kinds of matrices, scaffolds and hydrogel for both 3D and 2D cell culture. In this way, we are helping researchers to grow cells in more natural environments and mimic more closely the in vivo situation. We also offer a wide range of cell research products.
Where can readers find further information?
Please visit our website http://www.amsbio.com/microRNA-expression-lentivirus.aspx
For information on miRBase: http://www.mirbase.org/
About Maja Petkovic
Dr Maja Petkovic has been working with lentivirus products for more than two years. She is product manager for custom services and oversees lentivirus and adenovirus portfolio and long mRNA synthesis service. She is also managing aptamer products, immunological research products, RNA amplification and isolation products, enzyme assays, stem cell products and lab reagents.
Dr Petkovic has completed her PhD studies at University of Zurich, Switzerland studying DNA repair and maintenance of genomic stability. After that she has moved for post-doctoral training to Imperial College London, where she studied the role of forkhead transcription factors in breast cancer.