Tissue RNA Purification (Isolation, Extraction) Kit (Part No. RNAExt)

Tissue RNA Purification Kit

Features:

• Simple and reliable RNA isolation method
  
• Applicable to Real-time PCR (RT-PCR)
• Great for northern blotting, nuclease protection assays, RNA amplification for microarray analysis, cDNA library preparation
• Stable yield
• 50 preps per kit

Tissue RNA Purification Kit Protocol Overview

Applications

• Real-time-PCR (RT-PCR)
• Real-time quantitative
• Northern blotting
• Nuclease protection assays
• RNA amplification for microarray analysis
• cDNA library preparation after poly(A)+ selection

Protocol:  Tissue RNA Isolation

Materials required, but not supplied.
Chloroform
Ethanol (96–100%)

• Wash Buffer RPI and Wash Buffer RW are supplied as a concentrate. Before using for the first time, add ethanol (96–100%, purity grade p.a.) as indicated on the bottle to obtain a working solution.
• Use sterile, disposable, and individually wrapped plasticware.
• Use only sterile, disposable RNase-free pipet tips and microcentrifuge tubes.
• Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. Change gloves frequently, particularly as the protocol progresses from crude extracts to more purified material.
• Always use proper microbiological aseptic techniques when working with RNA.

Sample Preparation

Tissues
Tissue from animal or plant (either fresh or frozen at -70°C until use) can be processed by freezing with liquid nitrogen and grinding into a fine powder using a mortar and pestle.  Homogenize tissue samples in 1 ml Solution RL per 50–100 mg tissue using a tissue homogenizer or rotor-stator.

1. Incubate at 15-30°C for 5 minutes, to lyse the nucleiprotein complex completely.
2. Centrifuge at 12,000 rpm for 5 min at 4°C, transfer the supernatant to a new Rnase-free     microcentrifuge tube. This step can eliminate protein, fat, polysaccharide, musle or plant fibre.
3. Add 200 μl chloroform, mix by vortexing for 15 seconds, incubate at room temperature for 3 minutes.
4. Centrifuge the sample at 12,000 rpm for 10 min at 4°C.
5. Note: After centrifugation, the mixture separates into a lower, yellow phenol–chloroform phase, an interphase, and a colorless upper aqueous phase which contains the RNA. Transfer of the colorless, upper phase containing the RNA to a new RNase–free tube.
6. Add an 0.5 volume of ethanol. Mix well, a visible precipitate may form after adding ethanol. Transfer the mixture to a spin column, centrifuge at 12,000 rpm for 30 seconds at 4°C, discard the flow-through.
7. Add 500 μl Wash Buffer RPI (check whether ethanol is added or not), Centrifuge at 12,000 rpm for 30 seconds at 4°C, discard the flow-through.
8. Add 500 μl Wash Buffer RW (check whether ethanol is added or not), incubate at room temperature for 1 minutes. Centrifuge at 12,000 rpm for 30 seconds at 4°C, discard the flow-through. Repeat this step again.
9. Centrifuge the column at 12,000 rpm for 2 min. Air dry the column.
10. Place the spin column in a clean 1.5 ml microcentrifuge tube (not provided), and pipet 30-100 μl Rnase-free water directly onto the membrane. Incubate at room temperature for 2 minutes, and then centrifuge for 2 min at 12,000 rpm to elute. The tube contains the purified RNA. Store the RNA at -70℃.