TITLE: Pancreatic Islet RNA Extraction
AUTHORS: Islet  and Pancreas Analysis Core

[DESCRIPTION]
This SOP defines the methods used by the Vanderbilt Diabetes Center Islet and Pancreas Analysis (IPA) Core for RNA extraction of pancreatic islets isolated from mouse or human tissue.

[BEFORE_START]
Prepare all working surfaces and equipment by spraying generously with RNase Zap!

[STEPS]
SECTION: Reagent preparation
1. RNAqueous Kit – Can be stored at 4 °C up to 6 months.
SECTION: Reagent preparation
2. DNase Kit – Can be stored at -20 °C up to 6 months.
SECTION: RNA extraction procedure
3. Pick at least 75 islets into an RNase-free microcentrifuge tube. Islets should be free of acinar tissue and other debris.

For more information on picking islets with regard to size, refer to step 10 of the Static Incubation protocol:
 

SECTION: RNA extraction procedure
4. Centrifuge islets for 3 min at 200 rcf, 4 °C.

SECTION: RNA extraction procedure
5. Remove supernatant and add 1 mL cold RNase-free PBS.
SECTION: RNA extraction procedure
6. Repeat steps 4-5 twice, for a total of 3 PBS washes.
SECTION: RNA extraction procedure
7. Remove supernatant, add 200 mL Lysis Solution, and vortex to mix.
 

SECTION: RNA extraction procedure
8. Turn heat block to 75 °C.

Pipet total required volume of Elution Solution for all samples into a microcentrifuge tube, cap tightly, and place in heat block. Use the table below to calculate volume needed per sample (each elution will be performed in two steps). 

 

SECTION: RNA extraction procedure
9. Remove DNase kit from -20°C to thaw at -80 Room temperature.
SECTION: RNA extraction procedure
10. Add 100 µL 100% ETOH to each lysate and vortex to mix. If lysates were previously frozen, continue vortexing until thawed.
SECTION: RNA extraction procedure
11. Prepare a Micro Filter Cartridge Assembly for each sample: insert a cartridge into a round-bottom microcentrifuge tube, and label both pieces.
SECTION: RNA extraction procedure
12. Pipet 150 µL of lysate/ETOH mixture into the cartridge. Centrifuge for 10 s at 18400 rcf to pass the lysate through the cartridge and bind the RNA to the filter.
SECTION: RNA extraction procedure
13. Repeat step 12 for the final 150 µL of lysate.
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14. Add 180 µL Wash Solution 1 to the cartridge. Centrifuge for 10 s at 18400 rcf.
SECTION: RNA extraction procedure
15. Remove cartridge from tube and discard contents. Replace cartridge back into tube.
SECTION: RNA extraction procedure
16. Add 180 µL Wash Solution 2/3 to the cartridge. Centrifuge for 10 s at 18400 rcf.
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17. Repeat step 16 for a total of two washes with Wash Solution 2/3.
SECTION: RNA extraction procedure
18. Centrifuge for 1 min at 21300 rcf to remove any residual liquid from the filter.
SECTION: RNA extraction procedure
19. Transfer cartridge to a new, prelabeled round-bottom microcentrifuge tube. The schematic below illustrates steps 20-22.

   
SECTION: RNA extraction procedure
20. Add the first volume of pre-heated Elution Solution to the cartridge filter. Centrifuge for 30 s at 18400 rcf.
SECTION: RNA extraction procedure
21. Repeat step 20 with the second aliquot of Elution Solution. Remove and discard filter cartridge.
SECTION: RNA extraction procedure
22. Label an RNase-free microcentrifuge tube and transfer the Elution Solution (now containing RNA) from the round-bottom microcentrifuge tube to the RNase-free tube.
SECTION: DNase treatment procedure
23. Add 5 µL DNase Buffer and 1 µL DNase to each RNA sample. Vortex gently to mix.
SECTION: DNase treatment procedure
24. Incubate samples in 37 °C water bath for 20 min.
SECTION: DNase treatment procedure
25. Add 5 µL Inactivation Reagent to each sample, and vortex gently to mix.
SECTION: DNase treatment procedure
26. Incubate samples at 25 °C for 2 min, vortexing gently after 1 minute of incubation, and then again after the incubation is complete.
SECTION: DNase treatment procedure
27. Centrifuge samples for 90 s at 21300 rcf.
SECTION: DNase treatment procedure
28. Being careful not to disturb the pellet, pipet the supernatant from each sample into a new, prelabeled RNase-free tube.
SECTION: Measure RNA integrity and concentration
29. Assess the quality and concentration of RNA using the method of your choice. The IPA Core uses the services of the Vanderbilt University Medical Center VANTAGE Core.
SECTION: Reagent preparation
1.1.  Add 10.5 mL 100% ETOH to Wash Solution 1 bottle. Mark and date the bottle.
SECTION: Reagent preparation
1.2. Add 22.4 mL 100% ETOH to Wash Solution 2/3 bottle. Mark and date the bottle.
SECTION: Reagent preparation
2.1. Discard LCM Additive from DNase Kit.