Table 1.

RNA isolation methodology.

Method 1: Modified Trizol/RNeasy Hybrid
1.	Disrupt tissue with a 5 mm stainless steel bead using a TissueLyzer II instrument (Qiagen) in 350 µL of TRIzol at 50Hz for 5 minutes. Store the homogenate at room temperature for 5 minutes.
2.	Pass lysate through QIAshredder viscosity-reducing homogenization column (Qiagen) (Step tested during protocol refinement, see Figure 1).
3.	Add 10 µL of Proteinase K (Qiagen) and incubate at 55°C for 20 minutes (Step tested during protocol refinement, see Figure 1).
4.	Add chloroform to the homogenate (0.2 mL chloroform per 1 mL TRIzol) and shake vigorously for 20 seconds, then allow the sample to sit at room temperature for 2-3 minutes.
5.	Spin at 10,000 g for 18 minutes at 4˚C.
6.	Carefully remove aqueous phase (top) by aspiration and transfer to new sterile RNase-free tube (1.5 ml tube).
7.	Slowly add an equal volume of 100% ethanol, mix as needed.
8.	Load the sample (up to 700 µL) into an RNeasy column (Qiagen kit) seated in a collection tube and spin for 30 seconds at 8,000 g. Discard flow-through. Repeat as necessary.
9.	Add 700 µL buffer RW1 onto column and spin 30 seconds at 8,000 g. Discard flow-through.
10.	Transfer column into a new collection tube, add 500 µL buffer RPE and spin for 30 seconds at 8,000 x g. Discard flow-through. Ensure ethanol has been added to the RPE buffer before use.
11.	Add 500 μL buffer RPE and spin 2 minutes at 8,000 g. Discard flow-through.
12.	Spin the column for 1 minute at 8,000 g to get rid of any residual buffer in the column.
13.	Transfer the column to a new 1.5 ml collection tube and pipet 30-50 µL of RNase-free water directly onto the column membrane. Allow the sample to sit at room temperature for 1 - 2 minutes, and then spin 1 minute at 8,000 x g to elute RNA.
14.	Store RNA at -80°C until use.
Method 2: Modified RNAqueous
1.	Disrupt tissue with a 5 mm stainless steel bead using a TissueLyzer II instrument (Qiagen) in 500 µL of RNAqueous lysis buffer at 50 Hz for 5 minutes. Store the homogenate at room temperature for 5 minutes.
2.	Pass lysate through QIAshredder viscosity-reducing homogenization column (Qiagen) (Step tested during protocol refinement, see Figure 1).
3.	Add 10 µL of Proteinase K (Qiagen) and incubate at 55°C for 20 minutes (Step tested during protocol refinement, see Figure 1).
4.	Add equal volume of 64% ethanol.
5.	Load the sample into an RNAqueous column supplied with the kit.
6.	Wash column with 700 µL of Wash buffer #1.
7.	Wash column with 2 x 500 µL Wash buffer #2/3.
8.	Elute RNA with 40 µL pre-heated (75⁰C) elution solution.
9.	Store RNA at -80°C until use.