“Only 5% of total cellular RNA content is mRNA and is isolated by removing all other non-coding RNAs from the sample. The unique 3’-OH end of mRNA is utilized to separate it.”
Snippet:
- A gene is transcribed into mRNA which is then translated into a functional protein.
- mRNA comprises only 5% of total cellular RNA.
- mRNA has a long poly-A tail at the 3’-OH end which is its unique characteristic.
- Smaller non-coding RNA degrades mRNA to regulate transcriptional activity.
- A single mammalian cell has 10 to 30pg of total RNA.
DNA is a blueprint for life on earth, we already know why! But even though RNA is also a type of nucleic acid, it is functionally less known than DNA. Unlike DNA, which is the genetic material for us, RNA and its various types perform crucial functions for a cell.
Thus every possible RNA either coding or non-coding is as important as DNA. mRNA, tRNA, and rRNA are three popularly known while miRNA, siRNA and piRNA are other less-known RNAs present in a cell.
Broadly, we can understand various biological processes such as gene expression, gene regulation, transcriptional activities, post-transcriptional modifications and regulation of translation through mRNA studies. We need a pure, homogenous and adequate amount of RNA sample for that.
mRNA is particularly ‘the nucleic acid of interest’ because it’s the only one that can leave the nucleus and do the translation. The required molecules, proteins and enzymes for the translation are there; present in the cytoplasm of a cell.
The mRNA, transcribed from the DNA has only the coding exons come out from the nucleolar pores and manufacture a chain of amino acids. So when we only isolate the mRNA and reverse transcription into DNA, we can study the gene activity.
“The amount of mRNA present in a sample is directly proportional to the number of amino acid chains formed by that particular RNA and thereby the quantity of a gene that is expressed.” So the messenger RNA enables us to study gene expression.
The total cellular RNA is a homogenous mixture of all types of RNA and can’t be directly used for transcriptome analysis. We need only messenger RNA. So does it need a separate isolation process? How can we isolate only the mRNA? Let us find out.
In this article, I will explain the process and general principle of mRNA isolation, various separation techniques and optimization options.
Stay tuned.
Key Topics:
mRNA isolation from total cellular RNA:
There are various methods available to isolate mRNA from a total cellular RNA, however, for that first, we need pure RNA. Magnetic bead-based and spin-column-based mRNA isolation techniques are popularly known. No manual methods give an adequate amount of mRNA and are thus not advisable.
We need to purchase an mRNA isolation kit for this purpose. First, understand the basic and common mRNA isolation principle.
Principle:
The presence of a long poly-A tail or poly-adenylated tail at the 3’ end makes the complete mRNA and unique from others. The usual length of the poly-A tail is 100 to 300 nt (approx.) in most eukaryotes.
The process of poly-adenylation is explained in our previous article, you can read it here: What is a poly-A tail?
The process comprises four distinct steps:
- Hybridization of oligo(dT) primers with poly-A tail.
- Wash off unbounded RNAs.
- Removal of oligo (dT) Primers.
- Elution of mRNA (under low stringency conditions).
A particularly designed spin-column or magnetic beads with the poly- (dT) primers are allowed to bind with the poly-A tail of mRNA, followed by washing with a buffer that removes all other RNAs.
Only mRNA with poly-A tail and oligo-(dT) primers remain in the column or tube. The oligo-(dT) primers are, afterward, removed by using a specific buffer and mRNA is eluted in the elution buffer.
This is the broad principle of isolating the mRNA from the rest of the RNAs. Now let us see each technique in general.
Pre-preparation:
First, we need to extract total cellular RNA. The total RNA is extracted using the Trizol or PCI-based RNA isolation technique. Notedly, before processing, the quality, quantity and integrity of the RNA extraction should be assessed.
Before performing any of the techniques explained below, the RNA sample is heated at 55 to 65°C for 5 to 7 in order to denature the secondary structure of the RNA.
Cool the sample and allow it to hybridize with the oligo (dT) primer or beads.
Furthermore, prepare all the chemicals, reagents or resuspension advised by the manufacturer before initiating the isolation.
mRNA isolation by spin-column technology:
This solid-phase separation technique relies on the chemistry of hybridization between the poly (A) tail and the oligo (dT) primer-linked with the spin-column. The high-affinity oligo (dT) primers are linked with the silica of the column.
Under the action of the high salt concentration (buffer), the oligo (dT) column binds with the poly (A) tail of mRNA. Other nucleic acids will pass through the column but the mRNA will remain bounded there.
After removal of the unbounded RNAs, nucleic acid and other contaminants, a special low salt buffer is added to destabilize the mRNA poly (A): oligo (dT) complex. The mRNA is collected in the elution.
The present technique required repeated centrifugation steps. Care must be taken to avoid RNase contamination.
Related article: RNA: Structure, Types and Function.
mRNA isolation by magnetic beads:
The magnetic bead-based nucleic acid separation technique is a recently evolved high throughput, rapid and more accurate separation technique. The oligo (dT)- cellulose linked beads are used and available commercially.
The mRNA binds with the beads containing oligo (dT) and remains hybridized under a magnetic field. The supernatant is removed, and beads are collected and washed. The mRNA is eluted and collected in the elution buffer.
Note that companies provide a special separation buffer for separating the poly-A tail of mRNA from the oligo (dT).
Note:
- The salt solution of NaCl and Tris can be added to maintain the pH and allow effective poly-A and oligo (dT) hybridization under low stringent conditions.
- EDTA can be added to the RNA solution to prevent the action of RNase by blocking the metal ion binding sites.
mRNA isolation by chromatography:
Another excellent separation method chromatography is used in the isolation and purification of mRNA in recent times. Affinity chromatography with poly-(dT) coupled to the stationary phase binds with the mRNA present in the RNA sample and separates from the rest of the ribonucleic acids, protein and other impurities.
Usually, the poly- (dT) ligand containing chromatography column is available commercially. A high salt concentration buffer effectively provides hybridization between the poly-A tail of the mRNA and poly- (dT) ligands of the stationary phase.
The use of low salt concentration followed by hybridization removes other unbound nucleic acid present in the sample.
The sample is once or twice washed with the washing buffer and eluted in pH neutral elution buffer. Note that the low conductive and pH neutral elution buffer breaks bonds between the poly-A tail and poly- (dT) and only collects mRNA.
Post-processing of mRNA:
The quality and quantity of total extracted RNA are determined by spectroscopic analysis and gel electrophoresis to know DNA contamination, RNA degradation and total RNA content.
Evidently, traces of DNA also remain in the final mRNA, sometimes, however, is not a worse case for most of the uses but for PCR amplification and reverse transcription, DNase treatment is mandatory.
Manufacturers usually provide the DNase buffer with the mRNA kit. So no need to purchase it separately.
Suggestions:
- RNA is always at a risk by RNase, thus all the necessary precautions are taken to avoid the RNase interaction. This article will help you in this learning: 21 things to know for effective RNA isolation.
- Always use sterile and RNase-free utilities.
- Perform all the steps under a strict aseptic laminar hood.
- Strictly follow the manufacturer’s protocol.
- Prepare all the reagents using the DEPC water, use only nuclease-free DEPC water when required. To know more about what DEPC-treated water is and how to prepare and use it. Read the article by clicking the link.
Applications of isolated mRNA:
The isolated mRNA has pivotal significance in gene expression studies. It is used in mRNA sequencing, cDNA library preparation, microarray analysis, reverse transcription and transcriptomics studies.
Wrapping up:
RNA extraction itself is a tedious and painful process that needs high-end expertise and experience. mRNA isolation is important for gene expression studies. Thanks to the poly (A) tail present on the 3’-end, the process remained quite effective, every time.
So we can say the mRNA isolation technique is less ‘bone crushing’, still we need to take care of our total RNA.
Use safety regulation and RNA safety guidelines to protect the sensitive RNA sample from degradation. I hope this article will help you in your genetics learning.
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