Aug 01, 2025
Using reverse transcription digital droplet PCR to count rare cytoplasmic incompatibility factor transcripts
- Lore Van Vlaenderen1,
- J. Dylan Shropshire1
- 1Department of Biological Sciences, Lehigh University
- Shropshire Lab
Protocol Citation: Lore Van Vlaenderen, J. Dylan Shropshire 2025. Using reverse transcription digital droplet PCR to count rare cytoplasmic incompatibility factor transcripts. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbwdxygpk/v1
Manuscript citation:
Van Vlaenderen L, 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: June 30, 2025
Last Modified: August 01, 2025
Protocol Integer ID: 221333
Keywords: Wolbachia, Drosophila, ddPCR, density, titer, spike-in control, RT-ddPCR, cif mrna levels within individual insect tissue, cifb mrna from wmel wolbachia, cif mrna level, cifb mrna, drosophila melanogaster, reverse transcription digital droplet polymerase chain reaction, reverse transcription digital droplet pcr, rare cytoplasmic incompatibility factor transcripts this protocol, rare cytoplasmic incompatibility factor transcript, cif transcript, phenotypic data at unprecedented resolution, cif transcripts in duplex, reverse transcription, rna, using reverse transcription, digital droplet polymerase chain reaction, homology to cifa, phenotypic data, cifb sequence, individual insect tissue, digital droplet pcr
Funders Acknowledgements:
Lehigh University Faculty Research Grant
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Abstract
This protocol details four reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) assays to count cifA and cifB mRNA from wMel Wolbachia in Drosophila melanogaster. These assays count cif transcripts in duplex with either a synthetic spike-in RNA to normalize for technical variation or a D. melanogaster housekeeping gene to normalize for biological variation. With a limit of detection of about 1 cifA and 3 cifB copies per 20 μL reaction, the assays provide accurate and precise measurements across a wide dynamic range. We designed oligos with homology to cifA and cifB sequences from at least 33 Wolbachia strains, suggesting utility beyond wMel. These methods will allow researchers to measure cif mRNA levels within individual insect tissues, enabling efforts to pair molecular and phenotypic data at unprecedented resolutions.
Materials
Materials
- Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212
- ddPCR Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004
- ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007
- DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051
- DG8 gasketBio-Rad LaboratoriesCatalog #1864007
- Droplet generation oil for probesBio-Rad LaboratoriesCatalog #1863005
- Kimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155
- Microseal “B” Seal, AdhesiveBio-Rad LaboratoriesCatalog #MSB1001
- PCR plate, 96 wellBio-Rad LaboratoriesCatalog #1863005
- PCR plate, 96 well, ddPCRBio-Rad LaboratoriesCatalog #12001925
- PCR plate heat seal, foil, pierceableBio-Rad LaboratoriesCatalog #1814040
- 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
- Universal RNA Spike ITATAA Biocenter Catalog #RS25SI
- Water, molecular biology gradeMIDSCICatalog #KC248796
Note
Use high-quality plastics throughout this protocol. Low-quality pipette tips, for example, can result in droplet shearing, significantly reducing sample quality.
Oligos
- cifAwMelT1_dd forward primer: 5'-GGTCCTTGGAATAATTTGCGG-3'
- cifAwMelT1_dd reverse primer: 5'-TCAAACTCAGACTGTGGGC-3'
- cifAwMelT1_dd FAM-labelled probe: 5'-TTGCCACTTGATGGTTCTGGTGA-3'
- cifBwMelT1_dd forward primer: 5'-GCAAGGTACTAGAGCACAGG-3'
- cifBwMelT1_dd reverse primer: 5'-CACGAGCGTTGTTTCTACG-3'
- cifBwMelT1_dd FAM-labelled probe: 5'-AGGTGGTACTTCTACAGCACAAGG-3'
- B-Spectrin_dd forward primer: 5'-ATGACGACGGACATTTCGATTG-3'
- B-Spectrin_dd reverse primer: 5'-AACAGTCGGGAACTGGAGTTG-3'
- B-Spectrin_dd FAM-labelled probe: 5'-GGTCCTGGCAACGAGTACATCGAT-3'
- Universal RNA Spike ITATAA Biocenter Catalog #RS25SI
Note
The cifA- , cifB-, and β-Spec-ddPCR oligos should be used at primer:probe ratio of 900 nM:250 nM.
Spike oligos should be used at the provided concentration.
Equipment
- Centrifuge (Eppendorf, 5420)
- Centrifuge, for plates (Eppendorf, 5430R)
- Droplet generator (Bio-Rad, 1864002)
- Droplet reader (Bio-Rad, 1864003)
- Freezer (any)
- Pipette, 10 μL (Eppendorf, 4861000708)
- Pipette, 100 μL (Eppendorf, 4861000716)
- Pipette, 1000 μL (Eppendorf, 4861000732)
- QX Manager Software
- Rubber Brayer Roller, 4 inch (Amazon, B07YDNKSH6)
- Thermal PCR plate sealer (Bio-Rad, 1814000)
- Thermal Cycler (Bio-Rad, 1841100)
- Vortex mixer (MI0101002, Four E's Scientific)
Protocol materials
Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212
ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
Water, molecular biology gradeMIDSCICatalog #KC248796
ddPCR Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004
DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007
PCR plate, 96 well, ddPCRBio-Rad LaboratoriesCatalog #12001925
DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051
Universal RNA Spike ITATAA Biocenter Catalog #RS25SI
DG8 gasketBio-Rad LaboratoriesCatalog #1864007
Droplet generation oil for probesBio-Rad LaboratoriesCatalog #1863005
Kimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155
PCR plate heat seal, foil, pierceableBio-Rad LaboratoriesCatalog #1814040
Pipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
Microseal “B” Seal, AdhesiveBio-Rad LaboratoriesCatalog #MSB1001
PCR plate, 96 wellBio-Rad LaboratoriesCatalog #1863005
Troubleshooting
Before start
Collect and process samples to be analyzed via RT-ddPCR. We follow the protocol below to collect D. melanogaster testes, extract and purify RNA, eliminate contaminating gDNA, and synthesize cDNA through reverse transcription.
Protocol
CREATED BY
J. Dylan Shropshire
Clean workstation with 10 % (v/v) bleach and 70 % (v/v) ethanol.
Assemble PCR reaction
1h
Remove the following reagents from the freezer and allow to thaw On ice for 00:30:00 :
- ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- Water, molecular biology gradeMIDSCICatalog #KC248796
- Primers and probes, as appropriate
- cDNA samples
30m
Vortex reagents for 00:00:30 to mix.
30s
Centrifuge at 2000 rcf, Room temperature, 00:00:10 to bring contents to the bottom of each tube.
1m
Select one of the following assays.
Create a master mix in a Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 based on the described composition.
The final volume of each assay will be 20 µL , including 2 µL of DNA template.
Calculate a 10% excess.
5m
cifA/β-Spec and cifB/β-Spec ddPCR assay reaction composition:
- 10 µL ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- 6 µL Water, molecular biology gradeMIDSCICatalog #KC248796
- 1 µL cifA or cifB oligo mixture with FAM-labeled probe
- 1 µL β-Spec oligo mixture with HEX-labeled probe
- 2 µL cDNA template
Note
The cifA/β-Spec and cifB/β-Spec ddPCR assays are used to measure cifA and cifB transcript levels normalized to the transcription levels of a relatively stable host gene.
This assay is useful when:
- The number of Wolbachia transcripts per host transcription is more relevant than the total Wolbachia transcript count.
- Host cell numbers vary between treatment groups or samples.
cifA/spike and cifB/spike ddPCR assay reaction composition:
- 10 µL ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- 5.5 µL Water, molecular biology gradeMIDSCICatalog #KC248796
- 1 µL cifA or cifB oligo mixture with FAM-labeled probe
- 1 µL RNA spike-in control primer mixture
- 0.5 µL RNA spike-in control HEX-labeled probe
- 2 µL cDNA template
Note
The cifA/spike and cifB/spike ddPCR assays are used to measure cifA and cifB absolute abundance and correct for extraction and processing efficiency.
This assay is useful when:
- Technical variations, such as RNA extraction efficiency, can significantly impact results.
- The biological effect size is small and can be masked by technical noise.
- There is a need to back-calculate transcript abundance in the original sample.
Vortex the master mix for 00:00:30 to mix.
30s
Dispense 18 µL of the master mix into each well in a PCR plate, 96 wellBio-Rad LaboratoriesCatalog #1863005 .
10m
Add 2.0 µL DNA template to each reaction.
Add 2.0 µL Water, molecular biology gradeMIDSCICatalog #KC248796 to no template control reactions (NTCs)
10m
Use the Microseal “B” adhesive seal to seal the plate.
Use a roller to ensure the seal is tight.
Mix thoroughly by vortexing the plate for 00:00:10 .
1m
Centrifuge at 2204 rcf, Room temperature, 00:02:00 to bring contents to the bottom of each well.
Note
After centrifugation, check that there are no bubbles across the wells.
If there are bubbles, repeat the centrifugation.
2m
Generate droplets
9m 50s
Insert a DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007 into a DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051 .
Note
Check that the orientation of the cartridge is aligned properly in the cartridge holder.
Slide the edges of the holder toward the center to snap close.
15s
Peel away the adhesive seal to expose the sample.
Be gentle while removing the seal to avoid aerosolizing the samples.
By pipetting, transfer 19.5 µL of each reaction mix to the sample wells of a DG8 cartridge.
Avoid pipetting air bubbles into the DG8 cartridge.
Note
Check each sample in the DG8 cartridge for air bubbles.
Remove air bubbles by pipetting slowly using a different tip.
Air bubbles can interfere with droplet generation.
1m
By pipetting, add 70 µL Droplet generation oil for probesBio-Rad LaboratoriesCatalog #1863005 to the oil wells in the DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007 .
30s
Insert the DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051 into the droplet generator.
Close the lid.
Wait approximately00:02:00 for droplets to be generated
2m
While waiting, to prepare the next set of eight reactions.
4m
Open the droplet generator.
Replace the cartridge holder containing droplets with the cartridge holder that was prepared while waiting.
Close the lid to start droplet generation.
Remove the cartridge from the first cartridge holder and check for droplets.
By pipetting, transfer the droplet well (40 µL ) from the DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007 to a clean PCR plate, 96 well, ddPCRBio-Rad LaboratoriesCatalog #12001925 .
Note
If droplet generating is successful, the wells appear opaque.
Pipette slowly to avoid shearing the droplets.
2m
Once all the samples have been transferred, seal the PCR plate with a
PCR plate heat seal, foil, pierceableBio-Rad LaboratoriesCatalog #1814040 using a thermal PCR plate sealer set to heat the foil at 180 °C for 00:00:05 .
Safety information
When using a PX1 plate sealer, ensure the PCR plate is correctly aligned with the heat sealer. Always position the foil with the red line facing upward to prevent it from sticking to the machine and causing damage.
5s
Thermal Cycling
3h 1m
Transfer the sealed PCR plate containing droplets to a thermal cycler.
1m
Run the following program with a ramp rate of 1 °C per 00:00:01 and with a heated lid of 105 °C .
1 cycle of:
- 95 °C for 00:10:00 to activate polymerase and denature DNA.
50 cycles of:
- 94 °C for 00:01:00 to denature DNA.
- 60 °C for 00:02:00 to anneal primers and replicate amplicons.
1 cycle of:
- 98 °C for 00:10:00 to inactivate polymerase.
- 12 °C forever to store amplicons until use.
3h
Read droplets
1h
Transfer the assembled plate to the droplet reader, and secure it with the clamps in the machine.
Check that well A1 is in the top-left position and close the lid.
Safety information
If the plate is not secured properly, the sampling needle can stick to the lid and damage the instrument.
1m
Open the QX Manager software on the attached computer.
1m
If the droplet reader has not been used in the past week, it is important that the system be primed using the QX Manager software.
- Select the "System Utilities" tab.
- Select "Prime."
- Select "Yes" to begin.
- Select "OK" after priming is complete.
2m
Add a plate in the QX Manager software.
5m
Open the Plate Configuration window.
- Select the "Add Plate" tab.
- Select the plus icon beside "Add Plate."
- Select "Configure Plate" to open the plate configuration window with three tabs: "Plate Information," "Well Selection," and "Well Information."
Record general plate information.
- Select the "Plate Information" tab.
- Add a name for the plate and record it in your lab notebook.
- Define the supermix used in the reactions
- Define the file name and save the plate.
Identify the wells in the plate that contain droplets.
- Select the "Well Selection" tab.
- Select the wells that contain droplets.
- Click "Include Selected Wells."
Define the type of experiment and the contents of the wells.
- Select the "Well Information" tab.
- Click "Experiment Type" and select the appropriate type of experiment.
- Enter sample descriptions into the "Sample Description 1" through "Sample Description 4" fields.
- Select the appropriate "Sample Type" for each well.
- Click "Supermix" and select "Supermix for Probes."
- Click "Assay Type" and select "Single target per channel."
- Enter the target (e.g., cifA, cifB, Bspec, spike) in the "Target Name" field.
- Select the appropriate "Target Type" for each target.
- Click "Signal Ch1" and select "FAM."
- Click "Signal Ch2" and select "HEX" if reactions include Bspec or spike and "none" otherwise.
Select "Save" and "Start Run" to begin reading droplets
Note
The software performs a prerequisites check to ensure that the instrument is ready.
This will tell you if ddPCR Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004 needs to be added or if waste needs to be emptied.
Always add 50 mL of 10 % (v/v) bleach to the waste container after emptying.
30m
After the run is complete, save files associated with the run.
- Click "Save" to save the run.
- Select the "Data Analysis" tab.
- Click "Error Model" a select "Poisson" and "95%."
- Select the "Data Table" tab, ensure all columns are reported, and save it as a csv file.
- Select the "Run Information" tab and save the table.
- Select the "Reports" tab, click "Select All," and save the report as a PDF.
- Select the "1D Amplitude," "2D Amplitude," and "Concentration" tabs to save the plots associated with the run.
20m