RNA ISOLATION

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RNA ISOLATION (TRIZOL METHOD)

Extraction of Total High-Purity RNA from Cells or Tissues

1 Aim

To isolate and purify intact total RNA from biological samples (cells or tissues) utilizing the Guanidinium Thiocyanate-Phenol-Chloroform extraction method (commonly known as the TRIzol method).

2 Principle

RNA is notoriously unstable because RNases (enzymes that degrade RNA) are ubiquitous and incredibly tough to destroy. The TRIzol method overcomes this by immediately immersing the sample in a highly toxic, denaturing environment.

The Chemistry of Extraction:

• Guanidinium Thiocyanate: A powerful chaotropic agent that instantly unfolds and destroys all proteins, including highly resilient RNases.
• Phenol: Dissolves lipids and denatures proteins. It gives the TRIzol reagent its characteristic pink color.
• Chloroform (Phase Separation): When added to the mixture, it forces the homogenization to separate into an organic phase (bottom) and an aqueous phase (top), leaving RNA exclusively in the top layer.
• Isopropanol: Used to precipitate the RNA out of the isolated aqueous phase.

Fig 1: Phase separation after adding Chloroform and centrifuging. The RNA is safely isolated in the clear top layer.

3 Materials Required

Chemicals and Reagents

• Biological sample (fresh tissue or cell pellet)
• TRIzol Reagent (Toxic: Handle in fume hood)
• Chloroform
• 100% Isopropanol (Ice-cold)
• 75% Ethanol (prepared with RNase-free water)
• DEPC-treated or RNase-free water

Equipment

• Refrigerated Microcentrifuge (set to 4°C)
• Tissue Homogenizer or mortar/pestle
• Vortex mixer
• RNase-free microcentrifuge tubes & filtered pipette tips
• Ice bucket

The Golden Rule of RNA Work: Wear clean gloves at all times. Human skin is covered in RNases that will instantly degrade your sample if you touch the inside of a tube or a pipette tip!

4 Procedure Step-by-Step

Step 1: Cell Lysis & Homogenization

1. Take 50–100 mg of tissue or 1 × 10⁶ pelleted cells in an RNase-free tube.
2. Add 1 ml of TRIzol reagent. Work inside a fume hood.
3. Homogenize the sample completely using a mechanical homogenizer or by repeated pipetting.
4. Incubate the homogenate at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes.

Step 2: Phase Separation

1. Add 200 µl of Chloroform to the tube.
2. Cap tightly and shake vigorously by hand for 15 seconds. (Do not vortex, as this can shear genomic DNA and contaminate the aqueous phase).
3. Incubate for 2-3 minutes at room temperature.
4. Centrifuge at 12,000 × g for 15 minutes at 4°C.
5. Carefully transfer the upper, colorless aqueous phase to a fresh, sterile tube. Leave a little aqueous fluid behind rather than risking sucking up the white DNA interphase!

Step 3: RNA Precipitation

1. Add 500 µl of 100% Isopropanol to the aqueous phase.
2. Mix gently by inversion 5-6 times.
3. Incubate for 10 minutes at room temperature (or on ice for better yield).
4. Centrifuge at 12,000 × g for 10 minutes at 4°C. A tiny, gel-like white pellet of RNA will form at the bottom or side of the tube.

Step 4: RNA Washing & Dissolution

1. Carefully pour off or pipette out the supernatant, leaving the pellet untouched.
2. Add 1 ml of 75% Ethanol to wash the pellet (this removes precipitated salts).
3. Vortex briefly to dislodge the pellet, then centrifuge at 7,500 × g for 5 minutes at 4°C.
4. Discard the ethanol completely. Air-dry the pellet for 5–10 minutes. Do not let it over-dry or it will be impossible to dissolve.
5. Resuspend the RNA pellet in 20–50 µl of RNase-free water. Incubate at 55°C for 10 minutes to assist dissolution if necessary. Store immediately at -80°C.

5. Analysis & Quality Control

RNA must be checked for both Purity (via Spectrophotometer) and Integrity (via Agarose Gel).

Method	What to Look For	Ideal Result
NanoDrop (A260/280)	Checks for protein or phenol contamination.	Ratio of ~2.0
NanoDrop (A260/230)	Checks for salt, guanidinium, or alcohol carryover.	Ratio of 2.0 - 2.2
Agarose Gel (Integrity)	Checks if the RNA is physically degraded into pieces.	Two sharp bands (28S & 18S) with the top band twice as bright as the bottom (2:1 ratio). No smearing.

6. Troubleshooting Guide

Problem	Likely Cause & Solution
Low A260/280 Ratio (< 1.6)	Protein or Phenol contamination. You pipetted too close to the interphase/organic layer during Phase Separation. Be more careful next time!
Degraded RNA (Smear on gel)	RNase contamination during handling, or the tissue was not immediately frozen/processed after harvesting. Always wear fresh gloves.
Low Yield / RNA won't dissolve	The RNA pellet was over-dried (dried until clear instead of white/gel-like). Do not use a vacuum centrifuge to dry RNA.

7. Applications

• RT-qPCR: Measuring specific gene expression levels.
• RNA Sequencing (RNA-Seq): Whole transcriptome analysis.
• cDNA Synthesis: For cloning eukaryotic genes without introns.
• Northern Blotting: Detecting specific RNA sequences.

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🧠 Interactive Viva Quiz

Test your knowledge! Click on the questions below to reveal the correct answers.

1. Why is RNA inherently so much less stable than DNA?

✅ Answer: The 2'-OH group.

RNA has a hydroxyl (-OH) group on the 2' carbon of its ribose sugar (DNA lacks this, having just a Hydrogen). This 2'-OH group makes RNA highly susceptible to base-catalyzed hydrolysis and targeted attack by RNase enzymes.

2. Why do we add Chloroform during the TRIzol method?

✅ Answer: To induce phase separation.

Chloroform is highly non-polar. When mixed with the phenol-containing TRIzol and centrifuged, it forces the mixture to separate into a dense organic bottom phase (proteins/lipids) and an aqueous top phase containing the highly polar RNA.

3. When running your RNA on a gel, you see two very bright bands. What are they?

✅ Answer: 28S and 18S Ribosomal RNA (rRNA).

Over 80% of total cellular RNA is ribosomal RNA. Messenger RNA (mRNA) makes up only 1-5% and appears as a faint background smear. In highly intact RNA, the upper 28S band should be roughly twice as intense as the lower 18S band.

4. What is the purpose of washing the RNA pellet with 75% Ethanol?

✅ Answer: To remove precipitated salts.

RNA is precipitated using isopropanol, but salts from the guanidinium buffer also precipitate out. 75% ethanol contains enough water to dissolve and wash away these salts, but enough alcohol to keep the RNA safely pelleted.