In situ hybridization is done using 5’-fluorescein (FAM)-labeled 19mer (or sometimes 22mer) antisense oligonucleotides containing locked nucleic acid (LNA) and 2’-O-methyl-RNA (OME) moieties. LNA is placed at 5’and 3’ends and at every third base in the following configuration LooLooLooLooLooLooL (L=LNA, o=OME). These probes are labeled with FAM at their 5’ end for detection.
Probe design for miRNA is quite easy: Just take the antisense sequence of the mature
miRNA;. miRNA sequences can be easily found at mirbase: http://microrna.sanger.ac.uk/sequences/index.shtml
Probe design for mRNA is more demanding. We recommend to start by using the Dharmacon siDesign application: http://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx
This program is intended to design siRNA target sites and uses complex algorithms to determine accessible sites in the mRNA and also allows to do an automated BLAST search to select the most unique sites (i.e. not targeting a similar sequence stretch in another gene). Note that the purpose here is not to design a siRNA, but instead to use the algorithms to determine suitable unique target sites for the Superior Probes. The basic assumption is that good and accessible siRNA sites are also good and accessible sites for an antisense oligo. We take the suggested target sequences as provided by the siDesign program (19 mers) and then design our own antisense Superior Probes against these target sequences. You only need to know the accession number of your target mRNA to start the siDesign program.
All 19mer or 22 mer target sites should be thoroughly checked for uniqueness of the sequences using Nucleotide Blast (set for “somewhat similar sequences [blastn]”: http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome.
Hybridization temperature = Tm - 21°C. Probe Tm can be estimated at: http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
Rule of thumb: 60 °C is a good starting point for most Superior Probes. If you obtain a high signal: increase hybridization stringency.
Cell lines were maintained by serial passage in Dulbecco’s modified Eagle’s medium (DMEM). Cells were grown at 37°C and 5% CO2. Media were supplemented with 10% fetal calf serum, 2 mM L glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.
Cells were seeded on glass slides one day prior to the experiment. Cells growing on the glass slides were fixed in 4% buffered (pH=7) formalin for 10 minutes and permeabilized using 0.1% (v/v) Tween 20 in 4% buffered formalin for 5 minutes. Pre-hybridisation was done in hybridisation mix (50% (vol/vol) deionized formamide, 600 mM NaCl,10 mM HEPES buffer, pH 7.5, 1 mM EDTA, 5x Denhardt’s reagent and 200 mg/ml denatured herringsperm DNA (D6898, Sigma)) for 30 minutes. Hybridizations were done at 60 °C -65°C (depending on the probe) for 2-30 min in hybridization mix. Probes in hybridisation mix were heated at 95C for 10 minutes before use. The oligonucleotide concentration in the hybridization mix was 1-100 nM (must be determined empirically for each probe). After hybridization the tissue sections were washed consecutively for 5 min with 2x SSC, 0.5 xSSC and 0.2x SSC at the hybridisation temperature.
Hybridizations were done on 6-um sections of paraffin-embedded material. In brief: sections were deparaffinised using standard procedures, and boiled in citrate pH=6 (0.1M citric acid (MW=210.14 g/mol) + 0.1M trisodiumcitrate (MW=294.1 g/mol) in volume ratio’s 11.5:88.5) for 10 minutes (We find boiling is more reliable than Prot K treatment). Pre-hybridisation was done in hybridisation mix (50% (vol/vol) deionized formamide, 600 mM NaCl, 10 mM HEPES buffer, pH 7.5, 1 mM EDTA, 5x Denhardt’s reagent and 200 mg/ml denatured herring sperm DNA (D6898, Sigma)) for 30 minutes. Hybridizations were done at calculated Tm (often 60-65 °C, depending on the sequence of the Superior Probe) for 30 min in hybridization mix. The oligonucleotide concentration (i.e. the Superior Probe concentration) in the hybridization mix ranges between 1-100 nM (must be determined empirically for each probe). Superior Probes in hybridisation mix were heated at 95 °C for 10 minutes before use. After hybridization, the tissue sections were washed consecutively for 5 min each with 2 x SSC, 0.5 x SSC and 0.2 x SSC at the hybridisation temperature.
After 30 minutes of blocking in blocking buffer, the hybridization signal was detected using AP labelled FAB fragments (Roche 11 426 338 910) 1:500 dilution in blocking buffer for 1 hour. Blocking buffer was PBS / 1% w/v BSA / Tween20 0.02% / 1:100 sheep serum. The AP was visualized using Vector Alkaline Phosphatase Substrate kit III (Vector Labs SK-5300) and nuclear fast red was used as a nuclear counter stain. We prefer to use Vectamount (Vector Labs, H-5000 ) to mount our slides.
Note: this is a two day protocol and is different to the above mentioned short protocols. Hybridization time is still short but extra time is needed for wash steps.
Embryos were stored at -20 °C in 100% MeOH. Embryos were rehydrated through 75%, 50%, 25% MeOH in PBS containing 0.1% Tween-20 (PBST) and then 4 x 100% PBST washes – 5 mins each. Then the embryos were treated with Proteinase K (10ug/ml in PBST) for 10 min at 37 °C and post-fixed in 4% formaldehyde in PBST - 20 min at RT. Optional is a brief wash in water, block of endogenous APase activity in 0.1M triethanolamine and 2.5% acetic anhydride for 10 min at RT, brief wash in water, 5 x 5 min washes in PBST. Embryos were transferred to hybridization mix (HM+, (50% formamide, 5 x SSC, 0.1% Tween, 9.2mM citric acid,50 ug/ml heparin 500 ug/ml yeast RNA) for 2 hrs at 70 °C. Hybridisation was done in HM+ containing 10nM Superior Probe for 30-60 min at 70 °C.
Post-hybridization washes – 10mins for each of the following:
100% HM- (HM+ without heparin and yeast RNA)
75% HM-/25% 2 x SSCT (SSC containing 0.1% tween-20)
50% HM-/50% 2 x SSCT
25% HM-/75% 2 x SSCT
100% 2 x SSCT
Wash embryos for 6 x 15 min in 0.2 x SSCT. Embryos were then transferred to PBST through successive incubations for 2mins in:
75% 0.2 x SSCT/25% PBST
50% 0.2 x SSCT/50% PBST
25% 0.2 x SSCT/75% PBST
Block for 1 hour with 2% sheep serum/2mg/ml BSA in PBST. Incubate embryos overnight at 4 °C in blocking buffer containing anti-FAM-AP Fab fragments (1/2000)
Wash embryos 6 x 15min in PBST. Wash embryos 3 x 5min in staining buffer (Staining buffer: 100mM tris HCl pH 9.5, 50mM MgCl2, 100mM NaCl, 0.1% Tween 20). Staining was performed in buffer including 2.25ul/ml NBT (100mg/ml stock) and 3.5ul/ml BCIP (50mg/ml stock). Wash 3 x 5min in PBST. Stop reaction with 1mM EDTA in PBST for at least 1 x 5 min. Fix in 4% formaldehyde at RT for 20 min at RT. Wash 3 x 5min in PBST. Transfer embryos to 87% glycerol. Stereo microscopy is then performed.
Publications which included Superior Probes from the Amsterdam Medical Center:
1) Budde BS, Namavar Y, Barth PG, Poll-The BT, Nürnberg G, Becker C, van Ruissen F, Weterman MA, Fluiter K, te Beek ET, Aronica E, van der Knaap MS, Höhne W, Toliat MR, Crow YJ, Steinling M, Voit T, Roelenso F, Brussel W, Brockmann K, Kyllerman M, Boltshauser E, Hammersen G, Willemsen M, Basel-Vanagaite L, Krägeloh-Mann I, de Vries LS, Sztriha L, Muntoni F, Ferrie CD, Battini R, Hennekam RC, Grillo E, Beemer FA, Stoets LM, Wollnik B, Nürnberg P, Baas F. tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia. Nature Genetics 2008 Sep;40(9):1113-8. PMID: 18711368
2) Aronica E, Fluiter K, Iyer A, Zurolo E, Vreijling J, van Vliet EA, Baayen JC, Gorter JA. Expression pattern of miR-146a, an inflammation-associated microRNA, in experimental and human temporal lobe epilepsy. Eur. J. Neurosci. 2010 Mar;31(6):1100-7. Epub 2010 Feb 26.PMID: 20214679
3) Daphne de Launay, Jeroen Vreijling, Linda M. Hartkamp, Olga Karpus, Joana R.F. Abreu, Marjoein A. van Maanen, Marjolein E. Sanders, Aleksander M. Grabiec, Jorg Hamann, Henrik Oerum, Margriet J. Vervoordeldonk, Kees Fluiter, Paul P. Tak, and Kris A. Reedquist.
Silencing expression of Ras family GTPase homologues decreases inflammation and joint destruction in experimental arthritis. Am. J. Pathology 2010, in press