Materials and Reagents

Paraffin embedded specimen
100% xylene (Thermo Fisher Scientific, catalog number: 6601 )
100% ethanol
Protease K (Life Technologies, Ambion^®, catalog number: AM2546 )
Trizol (Life Technologies, Invitrogen™, catalog number: 15596-018 )
Chloroform (Sigma-Aldrich, catalog number: C2432 )
Glycogen (F. Hoffmann-La Roche, catalog number: 10901393001 )
Isopropyl alcohol (Isopropanol)
RNase-free water or TE buffer (USB, catalog number: 75834 ; Promega Corporation, catalog number: P1193 )
Tris-HCl (Life Technologies, Invitrogen™, catalog number: 15568-025 )
CaCl₂ (Sigma-Aldrich, catalog number: C5670 )
Sodium dodecyl sulfate (Life Technologies, Invitrogen™, catalog number: 15525-017 )
Commercial kit (Life Technologies, Ambion^®, Austin, TX)
Sodium dodecyl sulfate
Protease digestion buffer (see Recipes)

Equipment

Procedure

Cut 20 μm sections from the interior of the paraffin block using a microtome, to minimize the nucleic acid damaged by exposure to the atmosphere during storage (for recovery of miRNA ≥20 μm slices are recommended, otherwise the miRNA will be lost during deparaffin washes).
Place tissue slices into 1.5 ml siliconized tubes, and add 1 ml 100% xylene to the sample.
Incubate at 50 °C for 3 min to melt the paraffin. Centrifuge the sample for 1 min at maximum speed to pellet the tissue, then discard the xylene without disturbing the pellet. Repeat the xylene wash once.
Wash the pellet twice with 1 ml 100% ethanol and air dry.
Add 150 μl 1x protease K digestion buffer containing 500 μg/ml protease K to each sample, incubate at 55 °C for 3 h.
Add 1 ml Trizol to each sample, incubate at 15 to 30 °C for at least 5 min to dissociate nucleoprotein complexes. Add 0.2 ml of chloroform, vortex the tubes vigorously for 15 sec and incubate at 15 °C to 30 °C for 2 to 3 min. Centrifuge the samples at no more than 12,000 x g for 15 min at 4 °C.
Transfer the aqueous phase to a fresh tube, add 10 μg glycogen and mix. Precipitate the total RNA by mixing with 0.6 ml isopropyl alcohol, and put the tube at -20 °C for at least 1 h. Centrifuge at 12,000 x g for 10 min at 2-8 °C.
Wash the RNA pellet with 100% ethanol, briefly air-dry. Dissolve in RNase-free water or TE.

Notes

Most mature microRNAs and other small RNAs are intact, while most mRNA and other long RNAs are fragmented during formalin fixation. The yield is about 3 times higher than a commercial kit.
The RNA product is suitable for microRNA study or mRNA profiling by 3SEQ.

Recipes

Protease digestion buffer
20 mM Tris-HCl (pH 8.0)
1 mM CaCl₂
0.5% sodium dodecyl sulfate

Acknowledgments

This protocol was adapted from Ma et al. (2009).

References