The technology uses molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction without any enzymatic reaction. In brief, a probe library is made with two sequence-specific probes for each gene of interest...
- The first probe is a capture probe that is complementary to a particular target mRNA and contains also a short common sequence coupled to an affinity tag such as biotin.
- The second probe, the reporter probe, contains a second sequence complementary to mRNA and a color-coded tag (with >16000 codes) that provides the detection signal.
- All probes are mixed toghether with total RNA in a single hybridization reaction that results in the formatio of tripartite structures (probe#1-RNA-probe#2).
- After capture on the surface, an applied electric field extends and orients each tripartite structure in the same direction, and then the aligned frame is imaged and analyzed.
- Each target molecule of interest is identified by the color code generated by the ordered fluorescent segments (the barcode) and the level of expression is measured by counting the numbers of codes for each mRNA.
Geiss, G.K., Bumgarner, R.E., Birditt, B., Dahl, T., Dowidar, N., Dunaway, D.L., Fell, H.P., Ferree, S., George, R.D., Grogan, T., James, J.J., Maysuria, M., Mitton, J.D., Oliveri, P., Osborn, J.L., Peng, T., Ratcliffe, A.L., Webster, P.J., Davidson, E.H., Hood, L. (2008). Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnology, 26(3), 317-325. DOI: 10.1038/nbt1385
Are you introducing a new reporter technology? Have your lab developed a new reporter assay? Ask for a guest post or submit your paper (pdf) at firstname.lastname@example.org
Reportergene is now Ad-free and express solely the opinion of its editorial board.
Some days ago I was challenging the concept that a reporter need to be a protein. Imagine my surprise in reading the March issue of Nature Methods, where Romanov and colleagues from Attagene Inc, describe a novel method for simultaneously reporting on the activity of a large panel of transcription factors. Here the reporter is a synthethic sequence that isn't translated, but only transcribed, an example of RNA-based reporters. Moreover, the paper breaks the dogma 'one promoter - one reporter' by analyzing activities of multiple TFs with just 'one' reporter sequence.
The trick is that they cloned TFs binding sites in front of a reporter sequence with an enzymatic cleavage site in a separate position for each TF, so that after these constructs are transfected within a cell, digest of the reporter species produced spectrum of DNA fragments that mirrored TF activities profile.
Although CE is limited by a narrow dynamic range, and the assay is challenged by the management of up to 30 sequence transfection in the same cell culture, this novel technique offers a valuable alternative for TF activity fingerprinting that outclasses classic TF binding ELISAs for significance and elegance.
---/ citation /--- --- ---
Romanov, S., Medvedev, A., Gambarian, M., Poltoratskaya, N., Moeser, M., Medvedeva, L., Gambarian, M., Diatchenko, L., & Makarov, S. (2008). Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors Nature Methods, 5 (3), 253-260 DOI: 10.1038/nmeth.1186
Heavy metals like cadmium and mercury are "invisible" poisons that can accumulate and disrupt both metabolic and endocrine homeostasis of humans and livestock. While traditional analytical methods allow for highly accurate measurements of these metal concentrations, they commonly do not allow for time-dependent or location-specific in vivo measurements. As the uptake and distribution of this extremely toxic metal are not yet understood, highly sensitive and non-invasive methods are needed for its detection in a living organism. With the introduction of a cysteine residue at position 205, Chapleau and colleagues from the University of California, illustrate in Protein Science how the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensor for mercury:
- the mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH also in presence of metal binding agents like EDTA;
- these changes exhibit sigmoidal binding behavior with respect to the metal concentration with a detection limit in the low nanomolar range;
- time-resolved binding studies indicate rapid subsecond binding of the metal to protein;
- crystal structure and mass spectrometry further characterize this novel biosensor.
The aim of the authors is to provide a biosensor that, in presence of mercury, decrease (or anyway changes) its own fluorescence. Although (until now) the only "in vivo" application claimed in the title is the "GFP"-sensor expression in bacteria challenged with mercury 20 micromolar, the use of GFP as a mercury-specific noninvasive biosensor is a first example of a real-time biosensor that would enable the direct imaging of the uptake of heavy metals by a living cell, or (better) a living animal.
Chapleau, R.R., Blomberg, R., Ford, P.C., Sagermann, M. (2008). Design of a highly specific and noninvasive biosensor suitable for real-time in vivo imaging of mercury (II) uptake. Protein Science DOI: 10.1110/ps.073358908
«A reporter gene is one that codes for a protein easily detectable in a protein background»Such classic definition need to be revised when studying transcription factors (TFs): who really needs the protein (that may be unpredictably affected by irrelevant post-transcriptional mechanisms)? What are your feelings?