The overall goal of the following experiment is to detect the presence of mRNA molecules with single molecule specificity in single yeast cells. First, fix the yeast cells and formaldehyde using lyase containing buffer. Permeable the cells until most cells are not phased bright.
Next, incubate the perme cells with fluorescently tagged single stranded DNA Molecules then mount the cells on a plasma cleaned cover slip. Ultimately, the results obtained by fluorescence microscopy can detail the presence and localization of mRNA molecules in single cells. The main advantage of this technique over existing methods, such as gene expression, microarrays and northern blots, is that individual mRNAs can be visualized in single cells.
This method can help answer key questions in the transcription field. This includes quantifying the number of arm RNAs per cell in a population which can inform models of regulation at the promoter level. It can also be used to determine the localization and half lives of transcripts during the cell cycle.
In a plasma preen vacuum chamber position cover slips on the slides. Then place the vacuum chamber in a microwave and make sure it is sealed. Switch on the pump once the pump is started.
Turn on the vacuum now power on the microwave and stop five seconds after the plasma is visible. Then turn off the vacuum, followed by the pump. Pull out the vacuum chamber.
Then transfer the cover slips cleaned side up into 12. Well plates grow the yeast to an A 600 of around 0.1 to 0.2. In minimal media, 10 mils of cells yields enough for about 10 hybridization.
Add one 10th volume of 37%formaldehyde directly to the growth media and incubate at room temperature for 45 minutes. Centrifuge the cells at 3000 Gs for five minutes. Re suspend the pellet in one milliliter of buffer B and transfer to a micro centrifuge tube.
Wash twice with one milliliter of ice cold buffer B in a micro centrifuge tube. Centrifuge at 13, 000 RPM for one minute. Next, add one milliliter of sphero plating, buffer and incubate at 37 degrees Celsius for 15 minutes.
Monitor the cells microscopically every few minutes until most cells are black. Then wash twice with ice cold buffer. B.Spinning at a low speed of 3, 500 RPM.
Add one milliliter of 70%ethanol resus. Suspend the pellet gently and place overnight at four degrees Celsius or store indefinitely at minus 20 degrees Celsius. To prepare the hybridization solution, add one to three microliters of probe to 100 microliters of hybridization buffer.
Then vortex and centrifuge. We've imaged three different genes simultaneously using three different probe sets. Now centrifuge, the fixed yeast sample and aspirate off the wash buffer.
Add the hybridization solution and incubate in the dark with gentle shaking overnight at 37 degrees Celsius the next day. Treat clean cover lips with 150 microliters of 0.01%polylysine for five minutes. Then aspir and air dry.
The slides wash three times with dis distilled water and air dry after one wash with one XSSC Resus, suspend the cells in 150 microliters of 0.1 micrograms per milliliter. Freshly prepared dappy stain a aliquot the cell suspension onto the polylysine treated cover slips and incubate for 30 minutes. Next, thaw the prolonged gold mounting medium to room temperature.
Place three microliters onto a slide. Then add about 0.5 milliliters of ethanol to a cover slip. In the 12 well plate, remove the cover slip and air dry while holding with tweezers.
Place the cover slip cell side down onto mounting medium and let it harden several hours or overnight in the dark. Seal the edges with nail polish and proceed to imaging. Take a Zack of images around the reference point where the total distance is five micrometers with 0.2 micrometer size.
Repeat the imaging for each dye channel corresponding to each probe set. Then proceed as detailed in the accompanying text. To identify the cells by watershed segmentation, identify the spots using a radial gradient and measure the spots using a Gaussian fit.
Typically, histograms are computed from fish images and used to determine the number of mRNAs present in single cells. An important advantage of microscopy based RNA quantification is that one can obtain information on the localization of transcripts. For example, this fish uses single probes to identify mRNAs in single cells with an inducible CCB CBF one allele.
Because many mRNA molecules are present at the site of transcription, it is easy to identify the presence and location of transcription sites within the nucleus. The utilization of different dyes to label mRNAs of different genes facilitates quantification of multiple mRNA species in the same cells. In this experiment, yeast cells were incubated in the presence of alpha factor and sorbitol Using fish with the Stelara probes, the data show one cell responding only to the pheromone indicated by a red FUS one start site.
Simultaneously, another cell is predominantly responding to sorbitol, where the S TL one start site is labeled green. After watching this video, you should have a good understanding of how to label and visualize single mRNA molecules in yeast Once mastered. This technique can be performed properly in two days.
Remember to have a sufficiently dense concentration of cells. Dilute samples require more fields to be image and stored.
