A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

This method permits a transcriptomic analysis of a rare cell population from Drosophila male accessory gland, called the secondary cells, and it uncovers genes that make them critical for reproductive success. In the course of one day, this technique allows the sorting of secondary cells from different genotype and the extraction of the total RNA for subsequent qPCR or RNA sequencing. Sometimes projects are limited by the rarity of starting material.

This method can provide the basis for other groups facing the challenge of working with a rare cell population. Because the procedure needs to accumulate 40 glands, it's important to practice the dissections. Preparing the homemade pipette tips is also important, and so you should visualize it on the video how to make it and prepare in advance.

Before beginning the procedure, cut and flame round, wide, 200-microliter tips for handling the glands, and pass the tip opening of multiple 1, 000-microliter tips near a flame for less than one second to narrow the openings. Then, sort the modified tips from narrower to wider based on their speed of aspiration. For accessary gland dissection, place 20 to 25 male Drosophila in a glass dish on ice, and use forceps and a dissection microscope to remove the reproductive tract of one male fly.

Clear the accessory gland pair from all of the other tissues, including the testes and ejaculatory bulb, and transfer the accessory glands to a glass plate containing serum-supplemented medium at room temperature. When 20 pairs of accessory glands have been collected, wash the glands in a dish of PBS for one to two minutes at room temperature. At the end of the wash, transfer the glands to freshly prepared dissociation solution, and use sharp forceps under a dissecting microscope to firmly pinch the middle of a glandular lobe.

Using a second pair of forceps, cut the tissue with the sharp tip to separate the proximal region of the accessory gland and the ejaculatory duct from the portion of the gland containing secondary cells. Cut the glands so that the peptidases can reach the cells that are otherwise protected by the outer muscle layer and by the viscous seminal fluid in the lumen of the gland. Then, use a pre-wet, flame-rounded, wide, 200-microliter pipette tip to transfer the glands to a 1.5-milliliter tube.

For tissue digestion, place the tube in a 37-degree Celsius shaker for 60 minutes at 1, 000 rotations per minute. At the end of the incubation, stop the digestion with one milliliter of serum-supplemented medium, and use a pre-wet, flame-rounded, wide, 1, 000-microliter pipette tip to transfer the samples to a 24-well plate. Check the cells for their GFP expression under a fluorescent microscope.

Most of the gland tips should look intact, and a few secondary cells should be detached. Use a rounded, narrow, 1, 000-microliter pipette tip to gently triturate the cells three to five times to disrupt the accessory gland tissue. Then, use a very narrow, rounded, 1, 000-microliter pipette tip one to two times to individualize the cells.

Allow the cells to settle for 15 minutes. Confirm that the cells appear healthy under the microscope, and remove the excess medium. Then, transfer the cells into a new 1.5-milliliter tube, and rinse the wells with fresh medium to recover any remaining cells.

The Abd-B-GAL4 construct can be used in these experiments to label secondary but not main cells with GFP. In this study, both cell types were sorted from wild type and iab-6cocuD1 mutant accessory glands. This D1 mutant lacks the expression of Abd-B and MSA transcripts, which affect secondary cells development, morphology, and function.

After cell dissociation, FACS is used to select living cells based on DRAQ7, and GFP fluorescence level is used to isolate secondary cells and main cells. RNA is extracted from each cell population and adjusted to two nanograms per sample for subsequent RT-qPCR and sequencing. Quantification of the expression of specific genes by real-time quantitative PCR on wild type main and secondary cell extracts reveals that the secondary cell genes Rab19 and MSA are detected from only secondary cell extracts, while the main cell gene sex peptide is detected only from main cell samples.

Principal component analysis on RNA-sequencing data of three wild type and three mutant replicates reveals that the wild type replicates both cluster close together to each other and far from the mutant samples. Visualizing reads aligned to particular genes shows that housekeeping genes such as actin 5C, and the secondary cell-specific genes Rab19 and Dve are expressed in both genotypes. For MSA, however, the strong expression of the gene observed in wild type flies is lost in the mutant strains.

Obtaining enough living dissociated secondary cells is important for a successful procedure. Thus, verify the dissection and dissociation protocol in small-scale experiment. This method allows the sorting of both cell type of accessory gland from a small number of flies, enabling the study of how those cell react transcriptionally to multiple genetic or environmental conditions.

Male reproductive success is known to be affected by many things, including genotype, age, and mating status. The ease of this method should allow us to investigate how these factors affect accessory gland transcription.