How to Isolate mRNA from Total RNA

The oligo-dT/carrier combinations are mainly used in the purification of mRNA from total RNA by a technique known as affinity chromatography. There are two main methods to isolate mRNA from total RNA based on the type of cells; namely, direct method of mRNA isolation and minus method of mRNA isolation. Both methods are explained in this article.

Messenger RNA (mRNA) is a product of transcription, which is made up of a series of RNA nucleotides. It carries information in genes from the nucleus to the cytoplasm for the synthesis of proteins. RNA isolation from a particular cell line results in total RNA, which consists of rRNAs and tRNAs along with mRNAs. Hence, mRNA should be separated from the mixture of total RNA during the study of the transcriptome of the cell line. Unique properties of mRNA such as the presence of a poly(A) tail at the 3′ end of the mRNA molecule are used for the separation. Since oligo-dT molecules are complementary to the poly(A) tail, mRNA can be isolated from a mixture of total RNA.

Key Areas Covered

1. What is mRNA
     – Definition, Structure, Function
2. What is the Composition of Total RNA
    – mRNA, rRNA, tRNA
3. How to Isolate mRNA from Total RNA
    – Use of Oligo-dT/Carrier Molecules

Key Terms: Affinity Chromatography, Messenger RNA (mRNA), Minus Method, Oligo dT, Poly(A) tail, Total RNA

What is mRNA

The mRNA is a transcript of a protein-coding gene. It is produced inside the nucleus in eukaryotes and carries information for the production of a particular protein into the cytoplasm. It is translated into an amino acid sequence of the protein during translation, assisted by ribosomes. The primary transcript produced by transcription of eukaryotes is called the pre-mRNA.

Figure 1: mRNA Structure

During post-transcriptional modifications, a mature mRNA molecule is produced with several features such as 5′ cap and poly(A) tail. The total mRNA of a particular organism is known as its transcriptome.

What is the Composition of Total RNA

The result of RNA isolation is called the total RNA. It is composed of all three main types of RNA produced by a cell. They are mRNA, tRNA, and rRNA. Both tRNA and rRNA aid in translation. The size of eukaryotic mRNA is 0.5-20 kb. Transfer RNA (tRNA) brings corresponding amino acids during translation. It is 76-90 base pairs long and is composed of anticodon region, which is complementary to a particular codon on mRNA. Ribosomal RNA (rRNA) is a component of ribosomes. Some of the human rRNAs are 5 kb long.

Over 90% of the total RNA is composed of tRNA and rRNA. Only 1-5% of total RNA is mRNA. A typical mammalian cell may consist of approximately 500,000 mRNA molecules per cell. The amount of a particular type of mRNA can be 15-20,000 copies per cell.

How to Isolate mRNA from Total RNA

A number of molecular biology techniques such as cDNA library construction, sample preparation for microarray preparation, Northern blot analysis for weakly-expressed genes, etc. require High-quality mRNA. There are two main methods to isolate mRNA from total RNA based on the type of cells: direct method of mRNA isolation and minus method of mRNA isolation.

Direct Method of mRNA Isolation

The principle of the separation of mature mRNA from eukaryotic total RNA involves the affinity selection/affinity chromatography of polyadenylated mRNA with the use of oligo dT (oligodeoxthymidylate), which is complementary to the poly(A) tail. It is made possible by the absence of a poly(A) tail in the two other types of RNA: tRNA and rRNA. 

mRNA consists of 30-200 adenine nucleotides in its poly(A) tail. Affinity columns filled with oligo dT/carrier combination are used in the isolation of mRNA from total RNA. The oligo dT/carrier combination can be either cellulose-bound oligo dT, biotinylated oligo dT with streptavidin-coupled magnetic beads or oligo dT-coupled polystyrene-latex beads. All experimental conditions should be in RNase-free conditions to prevent the enzymatic breakdown of RNA.

Figure 2: Affinity Chromatography

Total RNA should be dissolved in a high salt buffer and heated briefly to 65-70 °C to disrupt secondary structures of RNA. The conditions for the isolation of RNA vary among commercially available kits for mRNA isolation. However, preparation of poly(A)-RNA or mRNA is composed of three steps.

1. Hybridization of poly(A)-RNA to oligo dT molecules connected to a carrier
2. Washing off of unbound RNA
3. Elution of poly(A)-RNA from oligo-dT/carrier combination under low stringency conditions

Minus Method of mRNA Isolation

Oligo dT-based purification of mRNA only yields mRNA with a poly(A) tail or mature eukaryotic mRNA. Hence, another method known as minus method can be used in the purification of mRNA that do not have a poly(A) tail. This method can be used in the isolation of mRNA from yeast and bacterial total RNA. It can also be used in the isolation of immature type of mRNA along with the mature mRNA from eukaryotic cells. It involves the transcriptome enrichment by depleting large ribosomal RNA from total RNA. The RiboMinus^TM Transcriptome Isolation kit from ThermoFisher Scientific uses three steps in the efficient purification of mRNA from yeast and bacterial total RNA.

1. Hybridization of total RNA with rRNA sequence-specific, 5′ biotin-labeled oligonucleotide probes
2. Removal of rRNA/5′-biotin-labeled probe complex along with the streptavidin-coated magnetic beads
3. Purification of impurities and elution of mRNA.

Conclusion

Total RNA is composed of the three main types of RNA: mRNA, rRNA, and tRNA. Purification of mRNA from total RNA is essential for the analysis of the transcriptome of a particular organism. The methods for purification are based on the type of mRNA. Eukaryotic mRNA, which contains a poly(A) tail, can be isolated by affinity chromatography with oligo dT. Immature eukaryotic mRNA and mRNA from yeast and bacterial cells can be isolated by depleting large rRNA from total RNA.

Reference:

1.“mRNA Extraction.” Thermo Fisher Scientific, Available here.
2.”RNA Extraction by RNA Type.”  Thermo Fisher Scientific, Available here.

Image Courtesy:

1. “Mature mRNA” (CC BY-SA 3.0) via Commons Wikimedia
2. “Affinity” By Jamit at English Wikibooks – Transferred from en.wikibooks to Commons by Adrignola using CommonsHelper (Public Domain) via Commons Wikimedia