Jan 10, 2022
Pancreatic Islet RNA Extraction
- 1Vanderbilt Diabetes Research Center
Protocol Citation: Islet and Pancreas Analysis Core 2022. Pancreatic Islet RNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.b2r9qd96
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 10, 2021
Last Modified: January 10, 2022
Protocol Integer ID: 55841
Abstract
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.
Materials
Reagents and supplies:
- P-200 Aerosol Resistant Tips (ART) (Fisher 212361)
- P-1000 Aerosol Resistant Tips (ART) (Fisher P-2079E)
- RNase-free microcentrifuge tubes (Ambion NC9302929)
- PBS (Gibco 14190144)
- RNase ZAP! (Ambion AM9780)
- 100% ACS grade ETOH
- P200 Micropipette (Eppendorf Research Plus or similar)
- P1000 Micropipette (Eppendorf Research Plus or similar)
Kits:
- RNAqueous Micro Kit with DNase kit (Ambion AM-1931)
Contains the following components:
- Micro Filter Cartridges
- Micro Tubes
- Wash Solution 1
- Wash Solution 2/3
- Lysis Solution
- Elution Solution
DNase Kit:
- LCM Additive
- DNase
- DNase Buffer
- DNase Inactivation Reagent
Equipment:
- Centrifuge with swinging bucket rotor
- Vortexer
- Heat block with microcentrifuge tube adapter
- -80°C freezer
- -20°C freezer
- Microcentrifuge
- Water or bead bath set to 37°C
Before start
Prepare all working surfaces and equipment by spraying generously with RNase Zap!
Reagent preparation
Reagent preparation
RNAqueous Kit – Can be stored at 4 °C up to 6 months.
Add 10.5 mL 100% ETOH to Wash Solution 1 bottle. Mark and date the bottle.
Add 22.4 mL 100% ETOH to Wash Solution 2/3 bottle. Mark and date the bottle.
DNase Kit – Can be stored at -20 °C up to 6 months.
Discard LCM Additive from DNase Kit.
RNA extraction procedure
RNA extraction procedure
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:
Protocol
NAME
Static Incubation of Pancreatic Islets
CREATED BY
Islet and Pancreas Analysis Core
Centrifuge islets for 00:03:00 at 200 rcf, 4°C .
Remove supernatant and add 1 mL cold RNase-free PBS.
Repeat steps 4-5 twice, for a total of 3 PBS washes.
Remove supernatant, add 200 mL Lysis Solution, and vortex to mix.
Note
At this point, lysates can be stored at -80 °C for future RNA extraction. Islets can also be stored dry, but it is recommended by the kit protocol to freeze in Lysis Solution.
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).
| A | B | C | D | E | F | |
| Sample IEQ | 75 islets | 100 islets | 150 islets | 200 islets | 250+ islets | |
| Elution steps | 10 μL + 10 μL | 15 μL + 10 μL | 15 μL + 15μL | 20 μL + 15 μL | 20 μL + 20 μL | |
| Total Elution Solution (μL) | 20 | 25 | 30 | 35 | 40 | |
| Total samples | ||||||
| Total Elution Solution for all samples (μL) |
Table 1: Recommended elution volumes. Copy and paste all cells above into an Excel sheet, then enter values into cells B4–F4. The total volume of Elution Solution required will be automatically returned in cell B6.
Remove DNase kit from -20°C to thaw at Room temperature .
Add 100 µL 100% ETOH to each lysate and vortex to mix. If lysates were previously frozen, continue vortexing until thawed.
Prepare a Micro Filter Cartridge Assembly for each sample: insert a cartridge into a round-bottom microcentrifuge tube, and label both pieces.
Pipet 150 µL of lysate/ETOH mixture into the cartridge. Centrifuge for 00:00:10 at 18400 rcf to pass the lysate through the cartridge and bind the RNA to the filter.
Repeat step 12 for the final 150 µL of lysate.
Add 180 µL Wash Solution 1 to the cartridge. Centrifuge for 00:00:10 at 18400 rcf .
Remove cartridge from tube and discard contents. Replace cartridge back into tube.
Add 180 µL Wash Solution 2/3 to the cartridge. Centrifuge for 00:00:10 at 18400 rcf .
Repeat step 16 for a total of two washes with Wash Solution 2/3.
Centrifuge for 00:01:00 at 21300 rcf to remove any residual liquid from the filter.
Transfer cartridge to a new, prelabeled round-bottom microcentrifuge tube. The schematic below illustrates steps 20-22.
Figure 1: RNA elution is performed in two steps, then RNA is transferred to a fresh microcentrifuge tube for DNase treatment.
Add the first volume of pre-heated Elution Solution to the cartridge filter. Centrifuge for 00:00:30 at 18400 rcf .
Repeat step 20 with the second aliquot of Elution Solution. Remove and discard filter cartridge.
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.
DNase treatment procedure
DNase treatment procedure
Add 5 µL DNase Buffer and 1 µL DNase to each RNA sample. Vortex gently to mix.
Incubate samples in 37 °C water bath for 00:20:00 .
Add 5 µL Inactivation Reagent to each sample, and vortex gently to mix.
Incubate samples at 25 °C for 00:02:00 , vortexing gently after 1 minute of incubation, and then again after the incubation is complete.
Centrifuge samples for 00:01:30 at 21300 rcf .
Being careful not to disturb the pellet, pipet the supernatant from each sample into a new, prelabeled RNase-free tube.
Measure RNA integrity and concentration
Measure RNA integrity and concentration
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.