Single-cell isolation allows genomic and transcriptomic analysis of one individual cell. It is also required to build a monoclonal cell line from rare cell isolation, that could be for example CRISPR-CAS9 gene edited cells. There are 3 popular methods: serial dilution, micro-manipulation and flow cytometry. None of these are easy or simple, and they often require expertise and experience. Fortunately, a new solution for everybody has come! It’s designed to isolate a single cell in a few seconds, and it’s (quite appropriately) called the Smart Aliquotor. [Read more…]
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…]
Accurate monitoring of genome transcription activities is of crucial interest for deciphering gene expression and for a better understanding of RNA biology. Over the past years, various experimental methods for RNA Pol II mapping density across the whole genome have been designed. Here, I’d like to offer a brief introduction to the human Native Elongating Transcript-Sequencing (NET-Seq) method.
Next generation sequencing has quickly become the preferred method over tiling arrays for most genomics and transcriptomics needs. The major exception has been the study of microRNAs, where highly sensitive probe arrays such as the 3D-Gene® miRNA profiling platform are still widely used. A large part of the reason for the persistence of array dominance in small RNA expression profiling is caused by the variability introduced in sequencing library prep protocols involving complicated hands-on PAGE purification steps.
The CleanTag™ Ligation Kit for Small RNA Library Preparation now allows users to remove the Gel Purification steps from their protocols and shift to more automated bead purification protocols. This is particularly important for cases when RNA quantity is limiting. Traditional small RNA library prep protocols will result in the formation of adapter dimers (similar to primer dimers) when RNA quantities are limiting, thus greatly reducing the number of usable reads. [Read more…]
As detailed in a previous post, chemically-modifying oligonucleotide adapters is an effective means to prevent adapter dimer formation during small RNA library prep. Just as primer dimers form when very little template DNA is used for PCR, adapter dimers can form with low starting concentrations of RNA. The CleanTag™ Ligation Kit for Small RNA Library Prep is a complete kit which is compatible with Illumina® technology that makes use of such modified adapters in optimized buffer conditions. The kit includes CleanTag™ chemically modified adapters that greatly reduce adapter dimer formation and is optimized for total RNA input from 1-1000 ng. [Read more…]
Epicentre® (an Illumina company) founded in 1987, has been bringing innovative enzymes and kits to the molecular biology, RNA biology, and sequencing communities for decades. In 2011, Epicentre® was acquired by Illumina®, Inc. largely for access to the Nextera™ technology, a great technological advance for DNASeq library prep, quickly rebranded as an Illumina® product. But what about other well-known Epicentre® brands?
“Toulouse may be the mRNA translation capital of France” declared third keynote speaker Jerry Pelletier at the symposium organized by Yvan Martineau, Stéphane Pyronnet et Julie Guillermet-Guibert of the CRCT ( Le Centre de recherche en Cancérologie de Toulouse) within the beautiful and modern Oncopole in Toulouse, France.
As detailed in a previous post, rRNA depletion with kits such as Ribo-zero greatly increases the number of usable reads in RNASeq studies, but this one-size-fits-all approach is not appropriate for some researchers. In eurkaryotic studies, researchers often find that poly(A) enrichment using the BioMag® SelectaPure mRNA System making use of BioMag® Oligo (dT)20 Particles is a cost-effective means to separate mRNA from rRNA and tRNA. Prokaryotic mRNAs are not polyadenylated however, so poly(A) enrichment is not a possibility for researchers working with microorganisms. Although there exist rRNA depletion kits for bacteria, the organism-to-organism variability in rRNA sequences may lead to suboptimal depletion in some species and even the unintended depletion of specific mRNAs.
An article published in PLoS ONE (DOI: 10.1371/journal.pone.0074286) has described a computer program specifically for the purpose of designing rRNA depletion probes for various organisms. Using the example of the 16S and 23S rRNAs of Mycobacterium smegmatis, the authors report improved mRNA integrity and abundance using this approach compared to using MICROBExpress™.
The organism-specific probe selection software is available for download here. Once designed, researchers can purchase Biotin-TEG DNA oligonucleotides and BioMag® Nuclease-Free Streptavidin Particles for efficient depletion of rRNA from their organism of choice.