RNA Isolation and Formaldehyde Gel Electrophoresis

Part A: TRIzol RNA Isolation

1. Homogenisation: Grind 150 mg of tissue into a fine powder using liquid nitrogen.
2. Lysis: Add 2 mL of TRIzol reagent to the powdered tissue.
3. Incubation: Incubate at room temperature for 5 to 10 minutes to dissociate nucleoprotein complexes.
4. Aliquot: Split the lysate equally into two sterile 1.5 mL microcentrifuge tubes (1 mL each).
5. Phase Separation: Add 200 µL of chloroform to each tube.
6. Agitation: Shake tubes vigorously by hand for 15 seconds.
7. Settling: Incubate at room temperature for 5 to 10 minutes.
8. Centrifugation I: Spin at 12,000 rpm for 8 minutes at 4 °C.
9. Precipitation: Transfer the clear upper aqueous phase to a clean tube.
10. Alcohol Addition: Add 500 µL of isopropanol to the collected aqueous phase.
11. Chilling: Incubate at -20 °C for 30 minutes to precipitate RNA.
12. Centrifugation II: Spin at 10,000 rpm for 8 minutes at 4 °C.
13. Washing: Discard supernatant and wash the pellet with 1 mL of 70% ethanol.
14. Drying: Centrifuge briefly, remove ethanol, and air-dry the pellet for 5–10 minutes.
15. Dissolution: Dissolve the RNA pellet in 20–30 µL of RNase-free water.

Part B: Formaldehyde Gel Electrophoresis

1. Gel Casting: Melt 1.2 g of Agarose in 90 mL of RNase-free water.
2. Buffer Addition: Cool to 60 °C, then add 10 mL of 10X FA Gel Buffer.
3. Pouring: Pour into a casting tray and allow it to solidify.
4. Equilibration: Submerge the gel in 1X FA Gel Running Buffer for at least 30 minutes.
5. Sample Prep: Mix RNA samples with 5X RNA Loading Buffer at a 4:1 ratio.
6. Denaturation: Heat samples at 65 °C for 3–5 minutes, then chill immediately on ice.
7. Running: Load samples and run the gel at 5–7 V/cm until tracking dye migrates 2/3 down.

Required Chemicals & Reagents

Also Read

Fluoroimmunoassay (FIA): Principle, Procedure and Applications

Core Isolation Reagents

• TRIzol Reagent: Lysis agent containing phenol and guanidinium thiocyanate.
• Chloroform: Induces phase separation (pure chloroform, no isoamyl alcohol added).
• Isopropanol: Precipitates RNA from the aqueous layer.
• Ethanol (70%): Prepared with DEPC-treated water for pellet washing.
• Liquid Nitrogen: Cryogenic grinding of tough tissue samples.
• RNase-free Water: Ultra-pure water treated with 0.1% DEPC and autoclaved.

Electrophoresis Reagents

• Agarose: Standard molecular biology grade for gel matrix matrix construction.
• MOPS (3-(N-morpholino)propanesulfonic acid): Buffering agent for the 10X FA buffer.
• Sodium Acetate: Ionic component for electrophoresis.
• EDTA (pH 8.0): Chelates divalent cations to inhibit remaining RNases.
• Formaldehyde (37%): Denatures RNA secondary structures during the run.
• Formamide: Powerful denaturant included in the loading buffer.
• Glycerol (100%): Provides sample density for well loading.
• Bromophenol Blue: Tracking dye for visual monitoring of migration.

Buffer and Gel Compounding

1. 10x FA Gel Buffer

• 200 mM MOPS
• 50 mM Sodium Acetate
• 10 mM EDTA

2. 1x FA Gel Running Buffer (1 Litre)

• 10X FA Gel Buffer: 100 mL
• 37% Formaldehyde: 20 mL
• RNase-free Water: 880 mL

3. 5x RNA Loading Buffer (12 mL Total Volume)

Also Read

Chemiluminescence Immunoassay (CLIA): Principle, Procedure and Applications

• 10X FA Gel Buffer: 4 µL
• 37% Formaldehyde: 720 µL
• Formamide: 1184 µL
• 500 mM EDTA (pH 8.0): 80 µL
• Bromophenol Blue solution: 16 µL
• 100% Glycerol: 2 µL
• RNase-free Water: 10 mL
• Storage: Stable for approximately 3 months at 4 °C.

 Required Precautions

RNase Decontamination

• Surface Cleansing: Wipe all pipettes, benches, and equipment with RNase-away or 70% ethanol.
• Consumables: Use only certified RNase-free, sterile plasticware and barrier filter pipette tips.
• Water Treatment: Treat all non-certified water with 0.1% DEPC for 12 hours, then autoclave.

Chemical Safety

• Fume Hood Use: Perform all steps involving TRIzol, Chloroform, and Formaldehyde inside a fume hood.
• Personal Protective Equipment: Wear a laboratory coat, safety goggles, and double-gloved Nitrile gloves.
• Phenol Burns: Keep PEG (Polyethylene glycol) nearby to treat accidental TRIzol skin contact.

Troubleshooting Guide

References

1. Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162(1), 156-159.
2. Rio, D. C., Ares, M., Hannon, G. J., & Nilsen, T. W. (2010). Purification of RNA using TRIzol reagent. Cold Spring Harbor Protocols, 2010(6), pdb-prot5439.
3. Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press.