RNA Extraction and Purification

• This is about the extraction and purification of nucleic acids, specifically RNA.

RNA Extraction

• Isolation of RNA molecules from a biological sample is achieved by removing contaminants, including proteins, DNA, and cellular debris.
• RNA extraction typically involves these steps: sample collection, cell lysis, phase separation, RNA precipitation, and RNA resuspension.

Steps in RNA Extraction

• Sample collection is the first step.
• Cell lysis involves disrupting cellular membranes.
• Phase separation is done via organic extraction method.
• RNA precipitation is the next step.
• RNA resuspension completes the process.

Sample Collection and Preservation

• Biological samples like cells, tissues, blood, and plants must be collected and stored properly to prevent RNA degradation.
• RNA stabilizing agents like RNAlater or freezing in liquid nitrogen is a tool for collection and preservation.

Cell Lysis and Homogenization

• Cells or tissues are lysed with detergents or chaotropic agents like guanidinium isothiocyanate.
• These agents disrupt cellular membranes and inactivate RNases.
• Mechanical disruption methods such as sonication, bead beating, or homogenization are used to help release RNA from the cells.

Phase Separation

• The most common method involves phenol-chloroform extraction, using reagents like TRIzol.
• Phenol-chloroform creates a biphasic separation consisting of an aqueous phase containing RNA, an interphase containing proteins, and an organic phase containing lipids and DNA.
• The aqueous phase, which contains the RNA, is collected.

RNA Precipitation

• RNA is precipitated by adding isopropanol or ethanol in the presence of salts like sodium acetate or lithium chloride.
• The RNA pellet is obtained by centrifugation.

RNA Resuspension

• The RNA pellet is air-dried and resuspended in RNase-free water or TE buffer to maintain stability.

Methods to Isolate RNA

• Guanidinium thiocyanate-based method.
• Spin column-based method.
• Combined guanidinium thiocyanate-column based method.

Guanidinium Thiocyanate Phenol-Chloroform Extraction

• The cell extract is extracted with phenol/chloroform at low pH.
• Guanidinium thiocyanate is used as a chaotropic agent used in protein degradation.
• Guanidinium thiocyanate disrupts cells, denatures proteins, and deactivates nucleases which stabilizes DNA, RNA, and protein.
• RNA is separated from DNA after extraction with acidic solution consisting of guanidinium thiocyanate.

Phenol-Chloroform Phase Separation

• Solution separates into an upper aqueous phase containing RNA and a lower organic phase containing DNA and protein under acidic conditions after centrifugation.

Spin Column Method

• The principle is selective RNA binding to a silica membrane.
• High-salt binding buffer, often containing ethanol, promotes RNA adsorption to the silica membrane inside the spin column.
• DNA, proteins, and other contaminants do not bind effectively under these conditions.
• A series of ethanol-based wash buffers remove proteins, DNA, and salts while removing impurities via wash steps.
• RNase-free water or low-salt buffer is used to elute purified RNA from the silica membrane during RNA elution.

Spin Column Method Procedure

• This is a faster method that avoids the use of toxic phenol and chloroform reagents.
• After homogenization in a guanidine-based denaturing buffer, the sample is passed through a spin column that binds the DNA.
• Alcohol or acetone is added to the flow-through with the RNA and protein, after which it is centrifuged through a second spin column that binds the RNA, leaving just protein.
• RNA is washed on-column and then eluted in water or Tris-EDTA buffer.

Guanidinium Thiocyanate-Column Based RNA Extraction

• Combines guanidinium thiocyanate-based RNA denaturation with silica membrane column purification for efficient and high-purity RNA isolation.
• Guanidinium thiocyanate effectively lyses cells and inactivates RNases, preventing RNA degradation.
• Spin column-based purification selectively binds RNA to silica membranes, removing contaminants like DNA, proteins, and lipids.

Isolation of Poly(A)+ RNA

• Poly(A)+ RNA is the template for protein translation, and most eukaryotic mRNAs carry tracts of poly(A) at their 3' termini.
• Methods developed for bacterial mRNA preparation focus on removing non-mRNAs rather than selecting mRNAs, since bacterial cells mostly lack poly-A tails.
• Two methods are commonly used in the selection of poly(A)+ RNA: column chromatography on oligo(dT) columns and batch chromatography.

Column Chromatography

• Used for the purification of large quantities greater than 25µg of poly(A)+ RNA isolated from mammalian cells.

Batch Chromatography

• Batch chromatography is the preferred method when working with small amounts, less than 50µg of total mammalian RNA.
• Batch chromatography can be used when many RNA samples are to be processed and is carried out with a fine grade of oligo(dT) cellulose.

RNA Purification

• RNA purification is essential to remove contaminants such as DNA, proteins, and salts after extraction.
• Silica Column-Based Purification spins columns containing silica membranes that selectively bind RNA.
• Washing steps remove impurities, and RNA is eluted using RNase-free water.
• Qiagen RNeasy Kits for Silica Column-Based Purification.
• Magnetic Bead-Based Purification binds RNA to silica-coated magnetic beads under specific buffer conditions.
• Dynabeads RNA purification system removes contaminants via magnetic separation and RNA is eluted in clean buffer.

Method to Assess RNA Quality and Integrity

• Purity is done via Spectrophotometry such as Nanodrop Analysis
• An A260/A280 ratio, a protein contamination check is optimal between 1.8-2.0.
• An A260/A230 ratio is used for salt and phenol contamination check is optimal is greater than 2.0.
• Integrity is done via Agarose Gel Electrophoresis or Bioanalyzer.
• Intact RNA shows clear 28S and 18S rRNA bands for eukaryotic RNA.
• An RNA Integrity Number (RIN) greater than 7 is considered good for sequencing.