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LNA28 /08.2005
 
 
In situ hybridisation on yeast
with Locked Nucleic Acid in situ hybridisation probes
 
Method prepared by Dr. Torben Heick, Department of Molecular Biology Aarhus University, Denmark

 
The protocol originates from the Singer laboratory (Long et al. (1997) Science 277, 383-387) and has been modified for yeast, Saccharomyces cerevisiae, cell cultures subjected to a heat-shock treatment. However, the outlined routines for fixation etc. should be applicable for yeast cell cultures in general.

Ref.: The protocol is modified from Thomsen et al. (2005) RNA, in press.
 

Fixation of yeast cells for RNA-FISH
  1. Cell cultures (10 mL) were grown at 25ēC to an OD600 of 0.1-0.3 and subjected to an instantaneous temperature-shift by addition of pre-heated media.

  2. After incubation at the desired temperature and time, e.g. 31ēC for 90 min, cells were fixed for 15 min at the experimental temperature by adding formaldehyde to a final concentration of 4%. This was followed by 30 min incubation at 20ēC.

  3. Fixed cells were pelleted by centrifugation and washed three times in 1 ml wash buffer (1.2M sorbitol, 0.1M KH2PO4/K2HPO4, pH 6.5), and subsequently resuspended in 200-500 μL wash buffer.


Mounting on class slides

  1. 10μl of the individual cell suspensions were plated on 14-well, 0.1% poly-L-lysine coated glass slides (Immuno-Cell Int.) and washed in wash buffer containing 1% β-merchaptoethanol before spheroplasting for 10-15 min at 30ēC in 10 μL oxalyticase solution (20mM vanadyl-ribonucleoside, 0.2% β-merchaptoethanol, 0.1 U/μL RNasin, 1.2M sorbitol, 0.1M KH2PO4/K2HPO4, pH 6.5, 0.1 mg/mL oxalyticase (Enzogenetics)). 
     
  2. Subsequently, cells were washed for 5 min at 20ēC; twice in wash buffer, once in (0.1M KH2PO4/K2HPO4, pH 6.5, 0.1% NP40) and once in (0.1M KH2PO4/K2HPO4 pH 6.5), and finally incubated with cold 70% ethanol for 15-30 min at -20ēC. 
Probe preparation
  1. Probe preparation was done by direct labelling of 10-20μg of a 5’-end amino modified Locked Nucleic Acid in situ hybridisation probes with 300μg of monofunctional Cy3 NHS-Ester (Amersham Pharmacia) in 0.1M NaHCO3/Na2CO3, pH 9.0 overnight at 25ēC in the dark. The probe was purified on a G-50 spin column and the concentration determined by OD measurement.

  2. For each well 100ng of purified probe was mixed with 10μg salmon sperm DNA and 10μg yeast tRNA, lyophilized and resuspended in 5μL solution I (80% formamide, 10mM NaHPO4 pH 7.0), denatured for 5 min at 95ēC and finally mixed with 5μL of solution II (4xSSC, 20mM Vanadyl-ribonucleoside, 4μg/μL BSA, 0.1U/μl RNasin). 
RNA-FISH
  1. Using a pipette, cells were drained for ethanol and washed in 10-20 μL (depending on well size) at 20ēC, twice for 5 min in 2xSSC and once for 10 min in 40% formamide/2xSSC, 0.1% Triton X-100, before overnight incubation at 37ēC with 10 μl of probe mix.

  2. Removal of probe solution was followed by washing steps in 10-20 μL volume: (i) twice in 40% formamide/2xSSC for 10 min at 37ēC, (ii) once in 2xSSC/0.1% Triton X-100 for 10 min at 20ēC, (iii) twice in 1xSSC for 10 min at 20ēC, and (iv) twice in 1xPBS for 5 min at 20ēC.

  3. Finally, 2.5μl of mounting solution (1xPBS pH 8.0, 80% glycerol, 0.1μg/ml DAPI) was applied to air-dried wells, which were subsequently covered with a cover slip and analysed by fluorescent microscopy.

 

NB: Optimisation of the standard conditions by increasing hybridization stringency conditions can be achieved by increasing the formamide concentration (normally 40%) in hybridization- and all appropriate wash-buffers to 50%, 60% and 70%, respectively.


Imaging
Images were acquired on an Olympus BX51 microscope equipped with cooled Olympus DP50 CCD camera and analysis software.
 
 


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