Jul 12, 2024
Simple protocol for combined extraction of exocrine secretion and RNA in small arthropods
- 1University of Graz
Protocol Citation: David Fröhlich, Dr. Guenther Raspotnig, Christoph Hahn, Bodner Michaela 2024. Simple protocol for combined extraction of exocrine secretion and RNA in small arthropods . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8mb7v5b/v1
Manuscript citation:
David Fröhlich, Michaela Bodner, Günther Raspotnig, Christoph Hahn, Simple protocol for combined extraction of exocrine secretions and RNA in small arthropods, Biology Methods and Protocols, Volume 9, Issue 1, 2024, bpae054, https://doi.org/10.1093/biomethods/bpae054
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: May 22, 2024
Last Modified: July 12, 2024
Protocol Integer ID: 100235
Keywords: biosynthetic pathways, chemical ecology, chemosystematics, differential expression analysis, gland secretion, oribatid oil glands, phylotranscriptomics, transcriptomics
Funders Acknowledgements:
Austrian Science Fund (FWF)
Grant ID: P33629-BBL
Austrian Science Fund (FWF)
Grant ID: P33840-B
Abstract
We here introduce a novel combination of different methods, namely gas chromatography-mass spectrometry and RNAseq. The described method can be used to extract exocrine chemical compounds and RNA from the same individual. Using this protocol, metobolites like defensive secretions, pheromones, surface protectans and others can be linked to RNA-profiling. The protocol should be applicable for the majority of arachnids, insects and other arthropods.
Materials
Equipment:
Microentrifuge
Homogenizer
Fume cupboard
Material:
Crushed ice
Methylene chloride
100% isopropanol
95% ethanol
Eppendorf tubes
GC-MS-vials
Tubes filled with beads (Beadbug‱ Prefilled 2ml Tubes with 1mm Triple-Pure-High-Impact Zirconium Beads)
RNA extraction kit: Promega ReliaPrep‱ RNA Miniprep System (other RNA extraction methods should work too)
Before start
This protocol has been tested for oribatid mites. Adaptations regarding your study organism might be necessary (e.g. accirding to organism size). Suitable modifications may include volume and types of solvent, duration of chemical extraction (adjustment recommended), additional preparation steps (e.g. using particular tissue only).
Preparation
Preparation
To prevent any kind of contamination (chemical substances, RNase,...) follow good laboratory practice. Wear gloves all the time.
Chemical extraction.
Put some crushed ice into a box and place it at your fume cupboard for chemical extraction.
For each sample, label two GC-MS vials with glass inlets and put them for cooling into the crushed ice.
Take some methylene chloride as solvent (other solvents should also be used, but were not tested).
Prepare all chemicals and equipment for RNA extraction. We used Promega ReliaPrep‱ RNA Miniprep System for RNA extraction. Steps according the manunfacturers protocol are marked with.*
We recommend to check the manufacturers protocol for more details before starting wth the extraction. Here you can find the manufacturers protocol:
reliaprep-rna-tissue-miniprep-system-protocol.pdf540KB
reliaprep-rna-tissue-miniprep-system-protocol.pdf540KB Before first use of the extraction kit prepare:*
-DNase I by adding nuclease-free water. Mix gently, do not vortex. Store at -20°C. Make aliquots to reduce freeze-thaw cylces.
-LBA + TG Buffer by adding 1-Thioglycerol to LBA Buffer. Mark bottle that you have performed this step.
-RNA Wash Solution by adding 95% ethanol (Not included in the kit). Mark bottle that you have performed this step.
-Column Wash Solution by adding 95% ethanol (Not included in the kit). Mark bottle that you have performed this step.
95% ethanol not included in the kit. Use volumes according to your kit size as mentioned in the manufacturers protocol p. 7f.
-Take 100% isopropanol for the extraction. (Not included in the kit)
Prepare 2 eppendorf-tubes 1.5ml and one Beadbug‱ Prefilled 2ml Tubes with 1mm Triple-Pure-High-Impact Zirconium Beads per sample. Label them accordingly to your samples. Prepare one additional eppendorf-tube too. (Not included in the kit)
Make sure a homogenizer and a centrifuge are ready.
Make sure all individuals of your organism of interest are ready.
Chemical extraction
Chemical extraction
15m
15m
Pipette 30 µL of methylene chlorid into one GC-MS-vial.
Transfer the individual(s) into the GC-MS-vials. Extract for00:15:00 . The vials shall be placed on the crushed ice during chemical extraction. Please note that we emphasis to shorten the extraction time. The ideal minimal extraction time may be species specific. Modify due to your knowledge on the study organism!
15m
Transfer the secretion-loaded solvent into a new vial. The extract can be stored at -20°C
If remnants of the solvent are visible, let them evaporate. Transfer the individual(s) into the bead-filled tubes for RNA extraction. Last remnants will evaporate during transfer.
RNA extraction
RNA extraction
4m
4m
Pipette 250 µL LBA + TG Buffer into the bead-filled tube. We use the manufacturers protocol with the tissue input range of ≤5mg. If your organism is larger (>5mg to 20mg) double the volume. We note every step where you have to adjust the volume.
Homogenize the samples (4m/s for 20 second, repeated after 20 seconds).
(After lysis in LBA + TG Buffer samples may be stored at –20°C to –70°C for up to three months.*)
Add 250 µL RNA Dilution buffer (RDB) (double for larger organisms; see step 9). Vortex for 10 seconds. Incubate for00:01:00 . Transfer the lysate into a new eppendorf-tube.
1m
Clear homogenates by centrifugation 10000 x g, Room temperature, 00:03:00 to pellet insoluble debris. Transfer the cleared lysates to clean tubes, taking care to avoid any pelleted debris.*
3m
Add 170 µL 100% isopropanol. For large samples (see step 9) use 340 µL .*
Wear clean gloves and open the packs of tubes and minicolumns carefully. Remove one ReliaPrep‱ Minicolumn, two Collection Tubes and one Elution Tube for each sample to be processed. Place the Collection Tubes in a microcentrifuge tube rack, and place the ReliaPrep‱ Minicolumn into a Collection Tube. Be sure to label all your tubes and minicolumns to maintain sample identity. Always wear gloves when handling the tubes and minicolumns.*
Transfer up to 700 µL of lysate to a ReliaPrep‱ Minicolumn and centrifuge 12000-14000 x g, 20-25°C, 00:01:00 . If your original homogenate LBA + TG volume was 500 µL , a second load step will be required. Remove the ReliaPrep‱ Minicolumn and discard the liquid in the Collection Tube. Place the ReliaPrep‱ Minicolumn back into the Collection tube. Repeat the centrifugation step.*
1m
Remove the ReliaPrep‱ Minicolumn, and discard the liquid in the Collection Tube. Place the ReliaPrep‱ Minicolumn back into the Collection Tube. Verify that the RNA Wash Solution has been diluted with ethanol. Add 500 µL of RNA Wash Solution to the ReliaPrep‱ Minicolumn. Centrifuge at 12000-14000 x g, 00:00:30 .*
30s
Empty the Collection Tube as before and place it in the microcentrifuge rack. In a sterile tube, prepare the DNase I incubation mix by combining (in this order) the following amount of each reagent per sample:
24 µL of Yellow Core Buffer
3 µL 0.09M MnCl2
3 µL of DNase I enzyme.
Mix by gentle pipetting; do not vortex. Prepare only the amount of DNase I incubation mix needed. Store the DNase I mix on ice while it is thawed. Apply 30 µL of this freshly prepared DNase I incubation mix directly to the membrane inside the column. Make sure that the solution is in direct contact with and thoroughly covering the membrane. The incubation solution is yellow to make this easier to see.
Note: Do not mix the Yellow Core Buffer and 0.09M MnCl2 prior to this step. The Yellow Core Buffer and 0.09M MnCl2 should be stored separately and mixed immediately prior to each set of RNA preparations.
Incubate for 00:15:00 at room temperature (+20 to +25°C).*
15m
After this incubation, add 200 µL of Column Wash Solution (verify that ethanol has been added) to the ReliaPrep‱ Minicolumn. Centrifuge at 12000-14000 x g, 00:00:15 . There is no need to empty the Collection Tube before the next step.*
15s
Add 500 µL of RNA Wash Solution (with ethanol added) and centrifuge at 12000-14000 x g, 00:00:30 . Empty wash solutions and discard the Collection Tube.*
30s
Place the ReliaPrep‱ Minicolumn into a new Collection Tube. Add300 µL of RNA Wash Solution (with ethanol added). Centrifuge at high speed for , 00:02:00 .*
2m
For each sample, remove one capped 1.5ml Elution Tube. Transfer the ReliaPrep‱ Minicolumn from the Collection Tube to the Elution Tube, and add15 µL Nuclease-Free Water to the membrane (double the volume for large samles [see step 9]). Be sure to completely cover the surface of the membrane with the water.*
Incubate for 00:01:00 .
1m
Place the ReliaPrep‱ Minicolumn in the centrifuge with the lids of the Elution Tubes facing out. Centrifuge at 12000-14000 x g, 00:01:00 . Remove the column and discard. Cap the Elution Tube containing the purified RNA and store at –70°C.*
Optional: You can repeat the elution process by again adding 15µl Nuclease-Free Water to the membrane, incubate for 00:01:00 and centrifuge at 12000-14000 x g, 00:01:00 again. (This has not been done with the samples in the original paper)
3m