Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

Our research focuses on understanding how cells sense, monitor, and regulate the multiple ribosomal DNA loci within their genome. Specifically, we aim to uncover the mechanisms that distinguish which rDNA loci are actively transcribed and how these loci are individually regulated. We have recently found that the amount of rDNA in a cell can change over time, and that this change alters which rDNA loci are transcribed.

We now understand that cells will change their rDNA transcription based on the amount of rDNA they have. This discovery leads us to ask how cells know how much RDA they have and how they determine which rDNA locus they will prefer to express. Our laboratory is now focused on understanding the mechanism cells use to distinguish between different rDNA loci and sense and regulate their activity.

Under a stereomicroscope, dissect Drosophila to isolate testes in RNase-free PBS. To begin, grasp the middle of the abdomen with one pair of forceps and the end of the abdomen with another pair to gently pull the animal apart. Using forceps, transfer the isolated testes from the dissecting dish into a 1.5-milliliter microfuge tube containing 0.5 milliliters of RNase-free PBS.

Aspirate the PBS and add one milliliter of fixation solution. Place the sample on a nutating shaker at room temperature for 30 minutes. After incubation, aspirate the fixation solution.

Then wash the sample two times with one milliliter of PBS for five minutes each on a nutating shaker. After aspirating the PBS, add one milliliter of RNase-free 70%ethanol and incubate the sample at four degrees Celsius overnight on a nutating shaker. Next, aspirate the ethanol and add one milliliter of wash buffer to the sample.

Incubate the sample at room temperature on a nutating shaker for three minutes. After that, let the tube rest upright for two minutes. Mix the probes and masks with the hybridization buffer to prepare a total volume of 100 microliters per sample as shown here.

Then add 100 microliters of hybridization solution to the sample after aspirating the wash buffer. Apply a transparent film to seal the edges of the tube. Wrap the tube in aluminum foil to protect it from light and incubate the sample in a 37 degree Celsius water bath for at least 24 hours.

Without aspirating the hybridization solution, add one milliliter of wash buffer to the sample. Incubate the sample in a 37 degree Celsius water bath for 30 minutes in the dark. Once the wash buffer is aspirated, add one milliliter of fresh wash buffer to the sample and incubate again.

At the end of the incubation, aspirate the wash buffer and add 50 microliters of mounting media to the sample. Under a dissecting stereomicroscope, use a pipette to transfer the sample onto a microscope slide. Using forceps, arrange the testes in a line on the microscope slide to facilitate sample identification during imaging.

Cover the sample with a cover slip and blot the edges of the cover slip with a tissue wipe to remove excess mounting media. Seal the edges of the cover slip with nail polish and leave them in the dark at room temperature for 10 minutes to allow the nail polish to dry. rRNA signals were detected in the nucleolus, appearing as DAPI pore regions within the nucleus, particularly in germ cells with large nuclei and nucleoli.

Faint, nonspecific signals were observed in the cytoplasm in samples lacking rRNA loci. In most cells, YrRNA signals were exclusively detected, indicating rRNA transcription from the YrDNA locus. Co-expression of X and YrRNA was observed in germ cells, with signals either merging into a single nucleus or separating into two nucleoli.