We need to find biomarkers for prognostic, diagnostic and personalised treatment development. Notably to fight cancers that affect tissues. Since biopsies are invasive, it’s better to look for biomarkers in body fluids. Indeed, a simple blood sample becomes a kind of ‘liquid biopsy’ to reveal tissues affections. For 13 years, increasing interest has been shown for miRNA as biomarkers and it will last for sure. The 2 main reasons are that they are major regulators of cell processes and they are released from tissues into the blood. They are major biomarker candidates in serum and plasma. Thus, these circulating miRNA (cmiRNA) are the best hope for modern medicine. Still, a lot of research has to be done to determine the specific signature for each pathology, and also depending on the patient background. Obviously, cmiRNA profiling is a key step and requires sensitive and reproducible method. Sequencing, qRT-PCR, several kind of microarrays… Let’s explore together what the best approach could be. [Read more…]
Piero Carninci’s group at RIKEN published an article last year demonstrating that small RNAs can possess a large variety of 5′ cap structures including a 2,7 dimethylguanosine modification and found multiple cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in small RNA pools.
This adds a further level of complication to small RNA sequencing library prep, as such protocols generally allow for sequencing only of RNAs with a 5′ monophosphate. In general, mature, functional microRNAs are thought to carry a simple 5′ monophosphate, and ligation kits such as the CleanTag™ Ligation Kit for Small RNA Library Preparation that use T4 RNA Ligase to fix oligonucleotides to the 5′ end of RNA will effectively oligocap this 5′-monophosphated RNA population. Carninci’s group used a variety of approaches including decapping with tobacco acid pyrophosphatase, CAGE, and immunoprecipitation with 2,2,7-trimethylguanosine to perform their study, but this isn’t the first time we have heard about cap structures on small RNAs.
We have known for quite some time that small RNAs can possess 5′ structures. The now discontinued ScriptMiner™ small-RNA–seq library prep kit from Epicentre® (an Illumina Company), for example, included protocols for removal of 5′ cap structures with Epicentre’s tobacco acid pyrophosphatase to allow capture of small 5′-capped and 5′-triphosphorylated RNAs in addition to 5′-monophosphate RNAs. Due to the discontinuation of tobacco acid pyrophosphatase, tebu-bio’s Decapping Pyrophosphohydrolase or CellScript’s Cap-Clip™ Acid Pyrophosphatase is now recommended to convert RNAs with diverse 5′ structures to 5′-monophosphate RNA for oligo-capping.
While next generation sequencing is quickly replacing array-based genomics/transcriptomics technologies, library prep for miRNA sequencing remains relatively complex. Due to the complexity of adapter-dimers and 5′ cap structures, array-based genome-wide miRNA expression analysis remains the first choice for many researchers.
The cap structures present on small RNAs appear to be either added during transcription or may be the result of a yet-to-be characterized mechanism for capping of RNA degradation products. An excellent resource for learning more about RNA 5′ structures is the Modomics website, which allows users to visualize various cap structures and gives the identify of the enzymes responsible for the modifications.
Here are some of tebu-bio’s top products and services for isolating and studying small RNAs:
- RNAzol® RT RNA Isolation Reagent – the most effective reagent for isolation of total RNA and small RNA from samples of various origins
- MasterPure™ RNA Purification Kits – for isolation of total RNA without columns and without dangerous solvents
- CleanTag™ Ligation Kit for Small RNA Library Prep – includes chemically modified adapters that greatly reduce adapter dimers
- iSWAB™ RNA – collection kit for RNA from buccal swabs or blood drops stabilizes donor samples at room temperature
- miRNA solutions – qPCR primers, 3’UTR target clones, precursor miRNA expression clones, miRNA inhibitors
- tebu-bio’s 3D-Gene® miRNA profiling platform – a full service array-based miRNA profiling service
- custom synthetic miRNAs and 2’-O-methylated oligonucleotides – tebu-bio can provide the most complex chemically-modified oligonucleotides
A recent study published in Nature Communications is the first to report how a mature microRNA (miR-223) actively controls the gene expression of a cell in which it was not originally transcribed. High Density Lypoprotein (HDL), commonly known as “good cholesterol”, is associated with reduced risk of cardiovascular disease. HDL particles are complex structures composed of many components, including proteins, bioactive lipids and… microRNAs. One of the known benefits of HDL is its ability to reduce inflammation that might integrate miRNA-driven pathways.
Recently, tebu-bio and Toray reached agreement for miRNA and mRNA profiling lab services. Already recognised as a European service provider in genomics, proteomics and cell-based assays, tebu-bio now brings European researchers access to Toray’s 3D-Gene® microRNA and mRNA profiling technology, offered through lab services.
During recent lab collaboration sessions, Toray’s 3D-gene® experts and tebu-bio’s lab staff shared their experiences and skills regarding miR and mRNA profiling technologies. A nice opportunity for me to ask Hideo Akiyama, PhD (Deputy General Manager – New Projects Development Division at Toray Industries, Inc.) 3 questions regarding the power of this “3D-gene® black resin” for microarray analysis.