Jul 31, 2025
Workflow to extract and process RNA from Drosophila testes with and without the endosymbiont Wolbachia
- Lore Van Vlaenderen1,
- J. Dylan Shropshire1
- 1Department of Biological Sciences, Lehigh University
- Shropshire Lab
Protocol Citation: Lore Van Vlaenderen, J. Dylan Shropshire 2025. Workflow to extract and process RNA from Drosophila testes with and without the endosymbiont Wolbachia. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1dqepvr2/v1
Manuscript citation:
Vlaenderen LV, Conner WR, Shropshire JD (2025) Counting cytoplasmic incompatibility factor mRNA using digital droplet PCR. bioRxiv. https://doi.org/10.1101/2025.07.30.667682.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 09, 2024
Last Modified: July 31, 2025
Protocol Integer ID: 114621
Keywords: RNA extraction, RNA purification, DNase treatment, cDNA synthesis, Drosophila, Wolbachia, TRIzol:Chloroform, phase separation, rna from drosophila testes, drosophila testes, rna from drosophila, drosophila mrna level, rna per testes, turbo dnase kit treatment, genomic dna contamination, contaminating genomic dna, purifying rna, processing rna, drosophila, dnase treatment, wolbachia cifa gene, rna, genomic dna, spectrin gene, gene, using digital droplet pcr, mrna level, digital droplet pcr, endosymbiont wolbachia this protocol
Funders Acknowledgements:
Lehigh University Faculty Research Grant
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Abstract
This protocol describes a complete workflow for extracting and processing RNA from Drosophila testes, including those harboring the endosymbiont Wolbachia. We begin by extracting and purifying RNA using TRIzol:chloroform phase separation, followed by a TURBO DNase Kit treatment to remove contaminating genomic DNA. cDNA is then synthesized using the SuperScript IV VILO Master Mix. Before DNase treatment, this method consistently yields approximately 52.5 ng of RNA per testes pair, with A260/A280 of ~1.94 and A260/A230 of ~1.45. Using digital droplet PCR (ddPCR) to quantify the Wolbachia cifA gene and the Drosophila β-spectrin gene, we confirmed that the DNase treatment completely eliminates genomic DNA contamination. This workflow is suitable for performing sensitive, accurate, and precise RT-ddPCR assays to measure both Wolbachia and Drosophila mRNA levels.
Guidelines
Important considerations:
- Unless otherwise stated, steps are at room temperature.
- Unless otherwise stated, use pipettes for liquid transfer.
- All reagents and materials should be certified RNase-free.
- Use only freshly opened consumables.
- Always wear a KN95 mask to protect samples from RNases.
- Speaking while performing extractions risks RNase contamination.
Materials
Materials
- Centrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212
- Centrifuge tubes, safe-lock, PCR clean, 2 mLEppendorfCatalog #022363344
- Chloroform, 99.8+%, ACS gradeThermo Fisher ScientificCatalog #032614.K2
- Conical tube, 50 mLOlympusCatalog #28-101
- Ethanol, 200 proofKOPTECCatalog #V1001
- Glycogen, UltraPureInvitrogen - Thermo FisherCatalog #10814010
- Homogenizing beads, 2.8 mm, ceramicVWR International (Avantor)Catalog #10158-554
- Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500
- Kimtech Kimwipe delicate task wipesKimberly-ClarkCatalog #34155
- Low EDTA TE (1X), pH 8.0Quality BiologicalCatalog #351-324-721
- Molecular Grade WaterMIDSCICatalog #786-73C
- Phosphate Buffered Saline, 10X SolutionFisher ScientificCatalog #BP3994
- Pipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
- Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
- Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
- Qubit RNA High Sensitivity (HS) Assay KitInvitrogenCatalog #Q32855
- RNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228
- SuperScript IV VILO Master MixInvitrogenCatalog #11756050
- TRIzol ReagentThermo Fisher ScientificCatalog #15596026
- Tubes, 0.2 mL, flat capThermo FisherCatalog #AB0620
- TURBO DNA-free™ KitThermo FisherCatalog #AM1907
- Universal RNA Spike ITATAA Biocenter Catalog #RS25SI
- Sodium Acetate TrihydrateFisher ScientificCatalog #S609500
Equipment
- Autoclave (Any)
- Bead-mill homogenizer (Benchmark Scientific, Bead Blaster 96)
- Centrifuge (Eppendorf, 5420)
- Centrifuge, refrigerated (Eppendorf, 5430-R)
- Chemical hood (Any)
- Drosophila Flypad (Flystuff, 59-119)
- Forceps, fine-tipped (Fine Science Tools, 11295-10)
- Freezer, -80C (Haier Biomedical, DW-30L1280TR)
- Graduated cylinder, 1 L (Any)
- Ice bucket (Any)
- Media bottle, 1 L, glass (Any)
- Mini centrifuge (VWR, 76269-064)
- NanoDrop Spectrophotometer (Thermo Scientific, ND-ONE-W)
- Petri dish, 35 mm, glass (Any)
- Pipette, 10 μL (Eppendorf, 4861000708)
- Pipette, 100 μL (Eppendorf, 4861000716)
- Pipette, 1000 μL (Eppendorf, 4861000732)
- Qubit 4 Fluorometer (Thermo Scientific, Q33238)
- Stereomicroscope (Zeiss, Stemi 305)
- Thermocycler (BioRad, C1000/S1000
- Thermomixer (Eppendorf, 5387000013)
- Vacuum chamber (Any)
- Vortex mixer (Any)
Preparations
- Dissecting dish: Add a thin layer of Sylgard 184Electron Microscopy SciencesCatalog #24236-10 to the bottom of a 35 mm glass petri dish. Remove bubbles using a vacuum chamber.
- 1x PBS: Using an autoclaved graduated cylinder, add to a 1 L media bottle: 100 mL Phosphate Buffered Saline, 10X SolutionFisher ScientificCatalog #BP3994 and 900 mL Molecular Grade WaterMIDSCICatalog #786-73C
- 75% ethanol: Using the graduations of a Conical tube, 50 mLOlympusCatalog #28-101 , mix: 30 mL Ethanol, 200 proofKOPTECCatalog #V1001 and 10 mL Molecular Grade WaterMIDSCICatalog #786-73C
Protocol materials
Kimtech Kimwipe delicate task wipesKimberly-ClarkCatalog #34155
RNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228
Universal RNA Spike ITATAA Biocenter Catalog #RS25SI
Low EDTA TE (1X), pH 8.0Quality BiologicalCatalog #351-324-721
Centrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212
Glycogen, UltraPureInvitrogen - Thermo FisherCatalog #10814010
Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500
Centrifuge tubes, safe-lock, PCR clean, 2 mLEppendorfCatalog #022363344
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Homogenizing beads, 2.8 mm, ceramicVWR International (Avantor)Catalog #10158-554
Qubit RNA High Sensitivity (HS) Assay KitInvitrogenCatalog #Q32855
Tubes, 0.2 mL, flat capThermo FisherCatalog #AB0620
TURBO DNA-free™ KitThermo Fisher ScientificCatalog #AM1907
Ethanol, 200 proofKOPTECCatalog #V1001
Sodium Acetate TrihydrateFisher ScientificCatalog #S609500
Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
SuperScript IV VILO Master MixInvitrogenCatalog #11756050
Molecular Grade WaterMIDSCICatalog #786-73C
Chloroform, 99.8+%, ACS gradeThermo Fisher ScientificCatalog #032614.K2
Sylgard 184Electron Microscopy SciencesCatalog #24236-10
Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
TURBO DNA-free™ KitThermo FisherCatalog #AM1907
Phosphate Buffered Saline, 10X SolutionFisher ScientificCatalog #BP3994
Pipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
Conical tube, 50 mLOlympusCatalog #28-101
Troubleshooting
Safety warnings
This protocol involves the use of various chemicals and reagents that require careful handling and strict adherence to safety guidelines to ensure safe laboratory practices. Please review the Material Safety Data Sheets (MSDS) for each reagent before beginning the protocol and take appropriate precautions. All steps should be performed while wearing gloves, a lab coat, and eye protection.
Testes dissection and sample collection
10m 15s
Thoroughly clean the working surface, stereomicroscope base, and fly pad with 10 % (v/v) bleach, 70 % (v/v) ethanol, and RNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Position an ice bucket to your dominant side of the stereomicroscope.
30s
Anesthetize males using CO2 and transfer them to a fly pad attached to a CO2 line.
Position the fly pad beside the stereomicroscope, on your non-dominant side.
1m
Fill a dissecting dish halfway with 1x PBS.
Position the dissecting dish under the optics of the stereomicroscope.
30s
Fold a Kimtech Kimwipe delicate task wipesKimberly-ClarkCatalog #34155 and position it behind the dissecting dish.
Saturate the Kimwipe with RNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
30s
Using fine-tipped forceps in your non-dominant hand, grasp a fly from the fly pad at the upper end of its abdomen.
Transfer the fly to the bottom of the dissecting dish, ensuring you do not release it.
5s
Using fine-tipped forceps in your dominant hand, grasp and gently pull the very tip of the fly's abdomen.
This action should release the abdominal contents, revealing digestive tissue, lipids, and testes.
Note
If the testes are not visible after pulling the abdomen tip, they may still be retained within the abdomen. Gently squeeze the abdomen with the forceps in your dominant hand, starting from the anterior and moving toward the posterior.
15s
Once the testes are clearly visible and separated from the fly, use the forceps in your non-dominant hand to transfer the fly to the RNase-Free-saturated Kimwipe positioned behind the dissection dish.
5s
Using both sets of forceps, carefully remove digestive tissues and other debris, transferring them to the RNase-Free-saturated Kimwipe.
10s
Using the forceps in your dominant hand, grasp the testes and transfer them to a Centrifuge tubes, safe-lock, PCR clean, 2 mLEppendorfCatalog #022363344 containing 800 µL of chilled TRIzol ReagentThermo Fisher ScientificCatalog #15596026 and three Homogenizing beads, 2.8 mm, ceramicVWR International (Avantor)Catalog #10158-554 .
Note
Placing the tissue directly into the TRIzol will cause it to stick to the forceps. Instead, place the tissue on the side of the tube above the TRIzol and then tilt the tube to immerse the tissue in the TRIzol.
10s
Cap the tube and store On ice .
Repeat steps to to collect more tissue for each sample or to collect additional samples. Repeat between sample groups.
Note
Dissection requires practice. It is reasonable to expect that a practiced user can reliably dissect a fly and transfer its testes to a sample tube within 45 seconds or less.
Transfer samples to a bead-mill homogenizer tube holder chilled to -20 °C .
1m
Homogenize the samples at 1500 rpm, 00:02:00 .
3m
Centrifuge the samples at approximately 15000 rpm, 00:00:30 to remove contents from the lid of the tube.
1m
Store samples at -80 °C until further processing.
RNA extraction and purification
2h 44m
Thoroughly clean the working surface and equipment surfaces with 10 % (v/v) bleach, 70 % (v/v) ethanol, and RNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Remove samples from the -80 °C freezer and allow the samples to thaw completely at Room temperature .
5m
Transfer samples to a bead-mill homogenizer tube holder chilled to -20 °C .
1m
Homogenize the samples at 1500 rpm, 00:02:00 .
3m
Centrifuge the samples at approximately 15000 rpm, 00:00:30 to remove contents from the lid of the tube.
1m
Allow samples to sit for 00:05:00 at Room temperature to ensure complete dissociation of nucleoprotein complexes.
5m
(Optional) Add 1 µL of Universal RNA Spike ITATAA Biocenter Catalog #RS25SI
pre-diluted to an appropriate concentration.
Note
Quantifying the abundance of synthetic spike-in RNA will enable the user to control for RNA processing variation between samples. We dilute the RNA Spike I 1:64 before adding to samples.
2m
(Optional) Prepare Spike I control sample:
- Add 24 µL Low EDTA TE (1X), pH 8.0Quality BiologicalCatalog #351-324-721 to a Centrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212 .
- Add 1 µL Universal RNA Spike ITATAA Biocenter Catalog #RS25SI pre-diluted to an appropriate concentration.
- Keep this control sample on ice throughout the extraction process.
2m
Thoroughly clean the working surface of the chemical hood with 10 % (v/v) bleach, 70 % (v/v) ethanol, and RNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Add 160 µL of Chloroform, 99.8+%, ACS gradeThermo Fisher ScientificCatalog #032614.K2 to each sample. Cap the tubes securely and shake vigorously for 00:00:15 or until the color is uniform throughout the sample.
Safety information
This step should be completed in a chemical hood.
Note
Shake immediately after adding chloroform. To process in bulk, place sample tubes in one rack and place another rack on top (sandwich formation) and shake.
1m
Incubate for 00:05:00 at Room temperature .
5m
Centrifuge at 12000 x g, 4°C, 00:15:00 to separate into three layers: a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase containing the RNA.
Note
The interphase is relatively small compared to the organic and aqueous phases and can be easily overlooked.
16m
Thoroughly clean the working surface and equipment surfaces withRNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Carefully remove 300 µL of the upper aqueous phase by pipetting at a slow speed (setting 2 on an Eppendorf Xplorer p1000) and transfer it to a new tube. Discard the old tube containing the organic phase.
Note
Be extremely careful not to pipette any of the organic phase (bottom red layer) or the interphase. Always keep the pipette tip at the top of the solution and do not aspirate the entire aqueous phase. Minimize tube movement after centrifugation to prevent phase disruption.
5m
Add 3 µL of Glycogen, UltraPureInvitrogen - Thermo FisherCatalog #10814010 to the aqueous phase.
Note
Glycogen acts as a carrier for RNA, improving precipitation efficiency.
1m
Add 400 µL of Isopropanol, 99.5%, molecular biology gradeThermo Fisher ScientificCatalog #327272500 to the aqueous phase and invert the samples 10 times to mix thoroughly.
2m
Incubate the sample for 00:10:00 at Room temperature .
10m
Centrifuge at 12000 x g, 4°C, 00:20:00 .
Note
The RNA pellet may be visible as a gel-like substance on the side or bottom of the tube, especially after centrifugation.
22m
(Optional): If the pellet is not visible or smaller than expected, invert the tubes and repeat to encourage pellet formation.
Thoroughly clean the working surface and equipment surfaces withRNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Pipette at a slow speed (setting 2 on an Eppendorf Xplorer p1000) to carefully remove all supernatant from the tube, leaving only the RNA pellet.
2m
Add 500 µL 75% ethanol to the RNA pellet.
2m
Vortex the sample for 00:00:10 .
1m
Centrifuge at 7500 x g, 4°C, 00:05:00 .
6m
Pipette at a slow speed (setting 2 on an Eppendorf Xplorer p1000) to carefully remove all supernatant from the tube, leaving only the RNA pellet.
2m
Repeat to three more times, for a total of four 75% ethanol washes.
33m
Centrifuge the samples at approximately 3200 x g, 00:00:15 and discard any remaining liquid by pipetting.
3m
Thoroughly clean the working surface and equipment surfaces withRNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Air-dry the RNA pellet for 00:10:00 by leaving the tube lid open. Avoid working directly over the samples to prevent contamination.
Note
A white pellet indicates salt contamination. A transparent pellet, once evaporated, suggests pure RNA. Do not allow the RNA to dry completely, as this significantly reduces solubility. Partially dissolved RNA samples will have an A260/280 ratio <1.6.
10m
Re-suspend the RNA pellet in 25 µL Low EDTA TE (1X), pH 8.0Quality BiologicalCatalog #351-324-721 by pipetting the solution up and down several times at a slow speed (setting 2 on an Eppendorf Xplorer p1000).
4m
Incubate at 60 °C for 00:10:00 at 0 rpm using the Thermomixer to facilitate dissolution.
10m
RNA clean up
1h 55m
Add 62.5 µL Ethanol, 200 proofKOPTECCatalog #V1001 and 2.5 µL 3M Sodium Acetate TrihydrateFisher ScientificCatalog #S609500 pH 5.2 to each sample.
Mix well by flicking the tubes and store the samples at -20°C overnight.
4m
Centrifuge at 21000 x g, 4°C, 00:30:00 .
31m
Thoroughly clean the working surface and equipment surfaces with RNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Pipette at a slow speed (setting 2 on an Eppendorf Xplorer p1000) to carefully remove all supernatant from the tube, leaving only the RNA pellet.
2m
Add 500 µL ice-cold 75% ethanol to the RNA pellet.
2m
Vortex the sample for 00:00:10 .
1m
Centrifuge at 7500 x g, 4°C, 00:05:00 .
6m
Pipette at a slow speed (setting 2 on an Eppendorf Xplorer p1000) to carefully remove all supernatant from the tube, leaving only the RNA pellet.
2m
Repeat to two more times, for a total of three 75% ethanol washes.
22m
Centrifuge the samples at approximately 3200 x g, 00:00:15 in a mini centrifuge and discard any remaining liquid by pipetting.
2m
Thoroughly clean the working surface and equipment surfaces withRNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Air-dry the RNA pellet for 00:10:00 by leaving the tube lid open. Avoid working directly over the samples to prevent contamination.
Note
A white pellet indicates salt contamination. A transparent pellet, once evaporated, suggests pure RNA. Do not allow the RNA to dry completely, as this significantly reduces solubility. Partially dissolved RNA samples will have an A260/280 ratio <1.6.
10m
Re-suspend the RNA pellet in 25 µL Low EDTA TE (1X), pH 8.0Quality BiologicalCatalog #351-324-721 by pipetting the solution up and down several times at a slow speed (setting 2 on an Eppendorf Xplorer p1000).
4m
Incubate at 60 °C for 00:10:00 at 0 rpm using the Thermomixer to facilitate dissolution.
10m
Quality control: Using a NanoDrop Spectrophotometer, measure nucleotide concentration (A260), protein contamination (A280/A260), and chemical contamination (A230/A260). Check the spectral curve to ensure that there is not a peak at A270, which would indicate TRIzol contamination.
15m
DNase treatment
52m 30s
Thoroughly clean the working surface and equipment surfaces withRNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Prepare a master mix containing 2 µL of TURBO DNase buffer and 1 µL TURBO DNase per sample from the TURBO DNA-free™ KitThermo Fisher ScientificCatalog #AM1907 kit. Prepare extra volume to account for pipetting errors.
4m
Mix the master mix by flicking the tube. Be gentle, but mix thoroughly.
30s
Centrifuge the master mix at approximately 3200 x g, 00:00:02 in a mini centrifuge to bring contents to the bottom of the tube.
1m
Distribute 3 µL of the DNase mixture into individual Tubes, 0.2 mL, flat capThermo FisherCatalog #AB0620 .
1m
Add 20 µL RNA to each reaction.
1m
Mix by flicking the tubes.
30s
Centrifuge the samples at approximately 3200 x g, 00:00:02 in a mini centrifuge to bring contents to the bottom of the tube.
1m
Incubate 37 °C for 00:30:00 in a thermocycler.
31m
Add an additional 1 µL or TURBO DNase to each sample.
Incubate 37 °C for 00:30:00 in a thermocycler.
Hold at 4 °C after incubation.
Add 2 µL inactivation reagent from the TURBO DNA-free™ KitThermo Fisher ScientificCatalog #AM1907 kit to each tube.
1m
Incubate for 00:05:00 at Room temperature . The inactivation reagent will settle during the incubation. Resuspend the inactivation reagent 2 to 3 times during the incubation by vigorously flicking the tubes.
Note
If room temperature is below 22 °C , incubate on a heat block at 25 °C .
5m
Centrifuge at 10000 x g, 00:01:30 to pellet the inactivation reagent.
2m 30s
Transfer 20 µL of supernatant to a Centrifuge tubes, safe-lock, PCR clean, 1.5 mLEppendorfCatalog #022363212 . Take care to avoid the white precipitate.
2m
Quality control: Perform a PCR reaction for 28s using the DNase-treated sample and run a gel. If DNA has been successfully removed, you should not be able to amplify this abundant host target. Use extracted DNA as a positive control. Include a no-template (water) control.
More details are available starting at step 13 in the following protocol:
Quality control: Measure RNA abundance using a Qubit 4 Fluorometer and Qubit RNA High Sensitivity (HS) Assay KitInvitrogenCatalog #Q32855 .
cDNA synthesis
30m
Thoroughly clean the working surface and equipment surfaces withRNase Free, Surface DecontaminantApexBio TechnologyCatalog #10-228 .
2m
Add the following, in order, to a Tubes, 0.2 mL, flat capThermo FisherCatalog #AB0620 :
- 4 µL SuperScript IV VILO from the SuperScript IV VILO Master MixInvitrogenCatalog #11756050 .
- 1 µL to 16 µL of RNA. Do not exceed 2500 ng per reaction.
- Water from the SuperScript IV VILO Master MixInvitrogenCatalog #11756050 to bring the final volume to 20 µL .
Mix by flicking the tube.
Note
Keep remaining RNA from each sample to use as controls for genomic DNA contamination.
2m
Centrifuge the SuperScript solution at approximately 3200 x g, 00:00:02 in a mini centrifuge to bring contents to the bottom of the tube.
1m
Run the following program on a thermocycler:
- Anneal primers - 25 °C for 00:10:00 .
- Reverse transcribe RNA - 50 °C for 00:10:00 .
- Inactivate enzyme - 85 °C for 00:05:00 .
- Hold at 4 °C .
25m
Store the cDNA at 4 °C for use within the next few days, -20 °C for use within the next few weeks, or -80 °C for longer-term storage.