In situ hybridisation
Each oligonucleotide labeled with fluorescent dyes is purified using a special HPLC procedure. Excess of dye as well as partial- and unmodified oligonucleotide is completely separated during this process. Quality control with Maldi mass spectrometry guarantees the identity of the oligonucleotide probes.
Depending on the experimental requirements several probe formats are available:
The classic probes with one fluorescent dye at the 5´-end of the oligonucleotide.
|Fam, Cyanine 3, Cyanine 5, Atto-dyes, Dyomics-dyes|
|Guaranteed yield||> 2 OD||> 5 OD||> 15 OD|
A nearly twofold increase in sensitivity may be achieved in in situ hybridization experiments (DOPE-FISH) by using double labelled probes. The biomers.net dopeProbes are labeled with an identical fluorophore at the 5´- and 3´-end of the oligonucleotide.
1. Double Labeling of Oligonucleotide Probes for Fluorescence in Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility. Stoecker K, Dorninger C, Daims H, Wagner M; Appl. Environ. Microbiol. (2010), 76, 922-926.
|3',5'-Fam, Cyanine 3, Cyanine 5, Tamra|
|Guaranteed yield||> 1 OD||> 3 OD||> 6 OD|
Highest sensitivity due to 4 labels per oligonucleotide. Nearly no influence on the hybridization properties as the dyes are coupled to the oligonucleotide backbone. Special probe-design prevents "self-quenching effects". The signal intensity of the hybridized probe nearly increases linear with the number of fluorophores per oligo; that means that tetraProbes may be two times brighter than dopeProbes and four times brighter than monoProbes under identical conditions.
|e.g. Fam, Atto 488|
|Guaranteed yield||> 1 OD||> 3 OD||> 6 OD|
Horseradish peroxidase labelled oligonucleotides
Oligonucleotides labelled with horseradish peroxidase (HRP), a 45 kDa large enzyme, are gaining more and more importance as probes for fluorescence in situ hybridisation (FISH) for detection of microorganisms. The combination of HRP probes and the tyramide signal amplification system (TSA) leads to 10- 20-fold increased signal intensities in comparison to fluorescein monolabelled probes. This "Catalysed Reporter Deposition - Fluorescent in situ hybridisation" (CARD-FISH) method represents an excellent tool for quantitative detection of microorganisms.
biomers.net has a long standing experience in synthesis and purification of high quality hrpProbes.
In a first step, an oligonucleotide, usually 18 - 25 bases long, is modified with an aminolink at the 5’-end. This primary amino function is coupled to a reactive bi-functional crosslinking reagent, resulting in an activated oligonucleotide, which can react in a further step with a free amino function of the horseradish peroxidase molecule. Thus, a stable covalent coupling is achieved. The appropriate choice of reaction conditions and excess of reagents leads to 1:1 connection between oligonucleotide and HRP.
Purification and isolation of the HRP oligonucleotide is best achieved by using polyacrylamide gel electrophoresis (PAGE).
biomers.net offers three synthesis scales of HRP oligos:
|Guaranteed yield||> 1 OD||> 2 OD||> 4 OD|
Other scales and final yields on request. For larger numbers of HRP-oligonucleotides per order please inquire for special offers.
smFISH - OligoPool 48
Oligonucleotides for single molecule in situ hybridisation (smFISH)
In gene expression analysis in tissue, in situ RNA detection is becoming increasingly important. But to detect a specific RNA with just a few copies in the cell, extremely sensitive methods are needed1.
In the so-called single molecule fluorescence in situ hybridisation (smFISH), single RNA molecules in fixed cells are detected using 48 short, single labelled oligonucleotide probes. Each probe is thereby marked with a fluorophore at its 3´-ending. By binding of all or many complementary oligo probes to different regions of the target, the desired mRNA is visible as a small fluorescent dot in the microscope2.
Since smFISH probes are also capable to bind to partially degraded RNA, the method is additionally suitable for fixed cells or embedded tissues. The large number of probes in the set ensures a high degree of sensitivity and specificity, so the likelihood of false-positive results is minimised3. If in individual cases a non-specific “off-target” binding occurs, only a weak fluorescence will be generated that is below the detection limit of specific mRNA binding. The use of different fluorescent dyes with clearly distinct emission spectra enables detection of several individual mRNA molecules in one cell. These so-called multiplex-imaging assays can create single cell gene expression profiles.
For detection of specific RNA in a cell, biomers.net offers a complete set of 24 or 48 single-labelled oligo probes. Each oligo pool is purified by HPLC and controlled by Maldi mass spectrometry.
|Modification||ABS [nm]||EM [nm]||Price category|
|Price category*||OligoPool 24||OligoPool 48|
|1||395,00 EUR / 299,00 GBP||459,00 EUR / 339,00 GBP|
|2||495,00 EUR / 390,00 GBP||595,00 EUR / 490,00 GBP|
|3||1290,00 EUR / 1161,00 GBP||1890,00 EUR / 1701,00 GBP|
Our excel order form for requests or orders can be download here.
For further information, please contact our customer support team at any time. We will be happy to support you.
Tel 0731-70 396 0 I firstname.lastname@example.org
* The prices are valid for an oligo pool with 24 or 48 oligonucleotide probes (18-25 bases) in scale S (guaranteed yield > 1 OD, 5 nmol pooled oligo) including HPLC purification and Maldi quality control. Prices are only valid for electronic orders (biomers.net online order form). VAT and shipping may apply. Upon request, we also offer oligonucleotide pools with 36 or 96 oligo probes. Additional conditions are according to our current price list.
1. Imaging individual mRNA molecules using multiple singly labeled probes. Ray A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S; Nature Methods (2008), Vol.5 No.10, 877-879.
2. Single Molecule Imaging of RNA in Situ. Batish M, Raj A, Tyagi S; Methods in Molecular Biology (2011), vol.714, DOI 10.1007/978-1-61779-005-8_1.
3. Single-molecule fluorescence in situ hybridization: Quantitative imaging of single RNA molecules. Kwon S; BMB Reports – Manuscript Draft, BMB-13-016 (2013), 65-72.
Links for probe design
For the design and application of probes in the field of in situ hybridisation we suggest the following links:
The SILVA rRNA database is a project of Max Planck Institute for Marine Microbiology, Bremen. SILVA database is the world´s largest collection for ribosomal RNA sequences for all three domains of life (Bacteria, Archaea and Eukarya) and thus it is one of the first addresses for taxonomic and phylogenetic questions. In addition to specially developed software for aligning, handling and analysis of sequence data, the website offers a comprehensive labor and troubleshooting collection for fluorescence in situ hybridisation.
Ribocon provides useful bioinformatic services and solutions for industry and academies in the area of environmental, clinical and molecular microbiology. Especially with microbial diversity analysis, phylogenetic studies and in the design of custom probes, Ribocon team is highly experienced.
The online server probeBase is maintained by the Department of Microbial Ecology at the University of Vienna. The online resource enables the targeted search of FISH and microarray probes and PCR primers by name, sequence or target organism. In addition, a detailed documentation of the probes is available.
The Department of Microbiology at the Technical University of Munich presents a collection of informative laboratory protocols and helpful links.