Ribonucleic Acid (RNA) is the major macromolecule essential for converting genetic codes into proteins through structures called ribosomes for performing cellular functions. The major types of RNA are Transfer RNA (tRNA), Messenger RNA (mRNA), and Ribosomal RNA (rRNA). It can be broken down into four nucleotide bases – adenine, cytosine, guanine, and uracil. The quality of RNA molecules is affected by factors like the storage conditions, tissue-processing protocols, and cellular stress response.
RNA has a single strand and contains ribose sugars, making it unstable and highly prone to degradation. Thus, special methods must be employed to preserve RNA in cells and tissues to keep it from degrading. This article describes some effective methods and RNA stabilizer for the preservation of RNA in cells and tissues.
Methods for Stabilizing Cells and Tissues
1. Snap Freezing
Snap freezing or flash freezing is the process of freezing little fragments of fresh tissues to preserve their RNA molecules. RNA in tissues has to be immersed in liquid nitrogen at a temperature of -196°C or in dry ice at -78°C. It can then be stored at a temperature of -80°C to keep it from melting. Though it comes with limitations, this method of stabilizing RNA is adequate and preserves the quality of the molecule.
If the ice or nitrogen in which the tissue was placed melts briefly, it can cause the RNA to degrade. Also, where there is no access to an efficient freezing facility, it becomes a problem. Transporting fresh and quality RNAs to a freezing facility has to be done with care. The tissue should be placed in liquid nitrogen or dry ice to avoid compromising RNA integrity.
To adequately isolate RNAs from tissues, the tissues have to be crushed in a mortar. Meanwhile, the RNAs have to be kept frozen while the crushing is being done, meaning liquid nitrogen will still be applied. The entire process is delicate and difficult. Dealing with numerous tissue samples becomes a problem as they can get contaminated when one mortar is used for more than one sample. Using separate mortars for every tissue sample is practically unrealistic when you havea lot of samples. An alternative is to allow the set to melt after the sample has been processed, then clean it up before starting the next extraction process.
Another limitation of this method is that powdered tissue samples can get thawed when transferred to a homogenization vessel. This would most likely result in clumps that hardly dissipates even when immersed in the lysis solution.
2. Formalin Fixation
Before going into formalin fixation as a method of stabilizing RNA in cells and tissues, we would first consider it as one of the sub-types of Protein Fixation. Fixation is the process of terminating biochemical reactions in tissues that have been separated from a living body to increase its mechanical strength and prevent it from breaking down. There are two types of fixation – the physical and chemical fixation. Physically, fixation can be performed by heating, microwaving, or cryo-preserving. Chemical fixation methods include immersion and perfusion fixation. The immersion fixation method employs the use of fixatives like formalin.
Formalin Fixation is a chemical method of fixing RNA Sequencing to tissues by creating cross-links between cellular proteins; and proteins and nucleic acids. The former cross-linking case makes it nearly impossible to extract Messenger Ribonucleic Acid (mRNA) from the tissue. After the formalin fixation step is the Paraffin Embedding step.These two steps make up the Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue.
During the paraffin embedding process, mono-methylol groups are added to the nucleotide bases (adenine, cytosine, guanine, and uracil) to form methylene bridges,which run between two groups of amino. One of the perks of employing formalin fixation for RNA stabilization is that it produces RNA with an acceptable integrity number during tissue extraction. However, it creates a chemical modification of the RNA when extracted. When proteins are cross-linked with nucleic acids, the mRNA gets firmly placed and becomes difficult to extract.
It is important to note that there are other fixatives like Methanol, Ethanol, Carnoy fixative, and Chloroform solution asides from formalin.
3. Chemical Preservatives
Two chemical preservatives that can be used as RNA Stabilizers are Aqueous sulfate solutions and TRIzol. Aqueous sulfate solution is a 100 ml solution with a pH of 5.2 made with Sodium Citrate, EDTA, and Ammonium Sulfate. Tissues can be preserved for weeks at room temperature using this solution. It can also keep tissues for extended periods at a temperature of -60°C.
TRIzol is a chemical reagent containing phenol and guanidine isothiocyanate. Although it is known to be effective for preserving the quality of RNAs (regardless of the quantity), TRIzol can irreparably break down tissues’ structure, making it unfit for histomorphological studies.
RNA is an essential component of every cell. It contains ribose nucleotides that are attached to form strands of different lengths. The ribose sugar has a chemically reactive hydroxyl group, which makes RNA unstable. This article explores some methods that can be used to stabilize RNA. The techniques discussed are snap freezing, Formalin Fixation – a subtype of Protein fixation – and chemical preservatives.