Pancreatic Islet RNA Extraction

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.

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:

Static Incubation of Pancreatic Islets

Centrifuge islets for 0h 3m 0s at 200rcf,4°C,0h 0m 0s.

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.

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)	=IF(SUM(B4:F4)>0,((B4B3)+(C4C3)+(D4D3)+(E4E3)+(F4*F3)+20),0)

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 -80Room 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 0h 0m 10s at 18400rcf,0h 0m 0s 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 0h 0m 10s at 18400rcf,0h 0m 0s.

Remove cartridge from tube and discard contents. Replace cartridge back into tube.

Add 180 µL Wash Solution 2/3 to the cartridge. Centrifuge for 0h 0m 10s at 18400rcf,0h 0m 0s.

Repeat step 16 for a total of two washes with Wash Solution 2/3 .

Centrifuge for 0h 1m 0s at 21300rcf,0h 0m 0s 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.

Add the first volume of pre-heated Elution Solution to the cartridge filter. Centrifuge for 0h 0m 30s at 18400rcf,0h 0m 0s.

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.

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 0h 20m 0s.

Add 5 µL Inactivation Reagent to each sample, and vortex gently to mix.

Incubate samples at 25°C for 0h 2m 0s, vortexing gently after 1 minute of incubation, and then again after the incubation is complete.

Centrifuge samples for 0h 1m 30s at 21300rcf,0h 0m 0s.

Being careful not to disturb the pellet, pipet the supernatant from each sample into a new, prelabeled RNase-free tube.

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.