Messenger RNA (mRNA) is a temporary copy of genetic information, serving as a blueprint for building proteins within cells. It carries instructions from DNA in the nucleus to ribosomes in the cytoplasm, where proteins are assembled. Isolating mRNA is a key technique in scientific research, providing insights into cellular processes and enabling biotechnological applications.
Why Isolate mRNA
Isolating mRNA is essential for studying gene expression, revealing which genes are active and to what extent in different conditions or cell types. This provides insights into cellular responses, disease mechanisms, and developmental processes.
It is also a preliminary step for creating complementary DNA (cDNA), a more stable DNA copy of mRNA, used for gene cloning, sequencing, or quantitative polymerase chain reaction (qPCR) to measure gene activity.
mRNA isolation has also been crucial in vaccine development, enabling the rapid creation of mRNA vaccines that deliver genetic instructions for producing viral proteins, prompting an immune response.
Additionally, isolated mRNA serves in diagnostic tools, aiding in the detection of specific gene activity patterns associated with various conditions.
Fundamental Considerations for Isolation
mRNA isolation is specialized due to RNA’s inherent properties. RNA is highly susceptible to degradation by ubiquitous ribonucleases (RNases). These enzymes rapidly break down RNA, requiring stringent sterile and RNase-free environments, including treated water and disposable plastics, to prevent degradation. Messenger RNA constitutes a small fraction (1-5%) of total cellular RNA; the vast majority is ribosomal RNA (rRNA) and transfer RNA (tRNA).
Most eukaryotic mRNA molecules have a poly-A tail, a distinguishing feature that allows for their selective isolation. This tail is a stretch of 100 to 300 adenine nucleotides attached to the 3′ end. The poly-A tail helps protect mRNA from degradation, assists in its transport, and plays a role in initiating protein synthesis. Its presence on mRNA, and absence on most other RNA types, forms the basis for many selective isolation techniques.
Key Steps in mRNA Isolation
Cell Lysis
The initial step in mRNA isolation involves breaking open cells to release their contents, including RNA, while simultaneously inhibiting RNases. This process, known as cell lysis or disruption, can be achieved through various mechanical or chemical methods. Mechanical methods include grinding tissues in liquid nitrogen, using bead mills, or homogenizers. Chemical lysis often employs solutions with strong denaturants like guanidinium thiocyanate (GITC) and phenol, which disrupt cell membranes and irreversibly inactivate RNases. The choice of lysis method depends on the sample type, with tougher tissues requiring more vigorous disruption.
Total RNA Extraction
After cell lysis, total RNA, DNA, and proteins are released. The next step separates RNA from these components.
Organic extraction, using phenol and chloroform, is a common method. Centrifugation separates this mixture into three layers: an upper aqueous phase (RNA), a middle interphase (denatured proteins and DNA), and a lower organic phase (lipids and other hydrophobic contaminants).
Alternatively, spin column-based kits use silica membranes that bind nucleic acids under specific salt conditions. Impurities are washed away, and the total RNA is then eluted.
mRNA Purification
After total RNA extraction, mRNA is purified from abundant ribosomal and transfer RNAs. This step exploits the poly-A tail of most eukaryotic mRNA. Oligo-dT affinity chromatography uses short deoxythymidine (oligo-dT) sequences immobilized on beads or columns. These oligo-dT sequences bind specifically to mRNA poly-A tails through complementary base pairing, while other RNA types without poly-A tails flow through. Magnetic beads coated with oligo-dT are also used, allowing for easy separation of mRNA-bound beads using a magnetic field.
Elution
The final stage of mRNA isolation is elution, releasing purified mRNA from the oligo-dT binding matrix into solution. This is achieved by reducing the buffer’s ionic strength, often by adding a low-salt solution or RNase-free water. The change in salt concentration disrupts hydrogen bonds between the mRNA’s poly-A tail and oligo-dT sequences, causing detachment from the beads or column. The eluted mRNA is then collected, ready for downstream applications like cDNA synthesis, sequencing, or gene expression analysis.
Ensuring Quality and Storage
After isolation, assessing mRNA quality is important for reliable downstream experimental results. Common methods include gel electrophoresis, which reveals RNA degradation by showing distinct bands for intact RNA and smears for degraded samples. Spectrophotometry quantifies RNA concentration and assesses purity by measuring absorbance ratios (e.g., A260/280 and A260/230), indicating protein or chemical contaminants. More advanced techniques like mass spectrometry can assess 5′ capping efficiency and poly(A) tail length.
Proper storage of isolated mRNA is important to prevent degradation and maintain stability. Due to its susceptibility to RNase activity and chemical degradation, mRNA is stored at ultra-low temperatures, such as -80°C, to inhibit enzymatic activity and molecular movement. For shorter periods, some mRNA products can be stored at -20°C or even 4°C, depending on the specific product and formulation. Storing mRNA in RNase-free water or specialized buffers in small aliquots helps avoid multiple freeze-thaw cycles, which can also contribute to degradation.