TITLE: Simple protocol for combined extraction of exocrine secretion and RNA in small arthropods 
AUTHORS: David Fröhlich, Bodner Michaela, Dr. Guenther Raspotnig, Christoph Hahn

[DESCRIPTION]
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.

[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).

[STEPS]
SECTION: Preparation
2. Chemical extraction.
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2.1. Put some crushed ice into a box and place it at your fume cupboard for chemical extraction.
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2.2. For each sample,  label two GC-MS vials with glass inlets and put them for cooling into the crushed ice.

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2.3. Take some methylene chloride as solvent (other solvents should also be used, but were not tested).
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3. 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:  

SECTION: Preparation
3.1. 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)
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3.2. 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) 
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3.3. Make sure a homogenizer and a centrifuge are ready.
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5. Pipette 30 µL of methylene chlorid into one GC-MS-vial.

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4. Make sure all individuals of your organism of interest are ready. 
SECTION: Chemical extraction
6. Transfer the individual(s) into the GC-MS-vials. Extract for15 min. 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!
SECTION: Chemical extraction
7. Transfer the secretion-loaded solvent into a new vial. The extract can be stored at -20°C
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8. 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.
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9. 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.

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10. 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.*)
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11. Add 250 µL RNA Dilution buffer (RDB) (double for larger organisms; see step 9). Vortex for 10 seconds. Incubate for1 min . Transfer the lysate into a new eppendorf-tube.

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12. Clear homogenates by centrifugation 10000 x g, 3 min to pellet insoluble debris. Transfer the cleared lysates to clean tubes, taking care to avoid any pelleted debris.*

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13. Add 170 µL 100% isopropanol. For large samples (see step 9) use 340 µL .*

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14. 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.*
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15. Transfer up to 700 µL of lysate to a ReliaPrep‱ Minicolumn and centrifuge 12000-14000 x g, 1 min, 20-25 °C . 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.*

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16. 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, 30 s.*

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17. 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 µLof 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 µLof 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 15 min at room temperature (+20 to +25°C).*

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18. After this incubation, add 200 µLof Column Wash Solution (verify that ethanol has been added) to the ReliaPrep‱ Minicolumn. Centrifuge at 12000-14000 x g, 15 s. There is no need to empty the Collection Tube before the next step.*

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19. Add 500 µLof RNA Wash Solution (with ethanol added) and centrifuge at 12000-14000 x g, 30 s. Empty wash solutions and discard the Collection Tube.*

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20. Place the ReliaPrep‱ Minicolumn into a new Collection Tube. Add300 µLof RNA Wash Solution (with ethanol added). Centrifuge at high speed for 2 min.*

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21. For each sample, remove one capped 1.5ml Elution Tube. Transfer the ReliaPrep‱ Minicolumn from the Collection Tube to the Elution Tube, and add15 µLNuclease-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 1 min. 


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22. Place the ReliaPrep‱ Minicolumn in the centrifuge with the lids of the Elution Tubes facing out. Centrifuge at 12000-14000 x g, 1 min. 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 1 minand centrifuge at 12000-14000 x g, 1 minagain. (This has not been done with the samples in the original paper)

SECTION: Preparation
1. To prevent any kind of contamination (chemical substances, RNase,...) follow good laboratory practice. Wear gloves all the time.