RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses. DNA extraction methods cannot be directly applied to RNA as RNA is structurally very different from DNA. RNA is single-stranded, while DNA is mostly double-stranded. It is often difficult to isolate intact RNA. RNases, a group of enzymes that degrade RNA molecules, are abundant in the environment, including on hands and on surfaces and it is difficult to completely remove/destroy RNases. RNA isolation, therefore, requires cautious handling of samples and good aseptic techniques. It is important to use only RNase-free solutions during the extraction, as well as RNase-free pipet tips and glassware.
RNA extraction methods are
• Guanidinium-acid-phenol Extraction
• Silica technology, glass fiber filters
• Density gradient centrifugation using cesium chloride or cesium trifluoroacetate
• Magnetic bead technology
• Lithium chloride and urea isolation
• Oligo(dt)-cellulose column chromatography
• Non-column poly (A)+ purification/isolation
The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues. TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. RNA remains only in the aqueous phase.
After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. But the DNA and proteins can recover by sequential separation after the removal of the aqueous phase. Precipitation with ethanol requires DNA from the interphase, and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA. This RNA can be used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection assay, and molecular cloning.
• Isopropyl alcohol
• 75% Ethanol
• RNase-free water or 0.5% SDS solution
The SDS solution must be prepared using DEPC-treated, autoclaved water. DEPC inactivates the RNases by the covalent modifications of the histidine residues.
Growth medium on the cells was discarded and cells were washed with ice-cold 1X PBS. The monolayer was then covered with 1 ml of l TRIzol and the cells were lysed and homogenized by repeated pipetting
2. Phase Separation
• The homogenized samples were incubated for 5 minutes at 15 to 30°C for the complete dissociation of nucleoprotein complexes.
• 0.2 ml of chloroform per 0.75 ml of TRIZOL Reagent was added. The tubes were shaken vigorously by hand for 15 seconds and incubated them at 15 to 30°C for 2 minutes.
• The samples were centrifuged for 15 minutes at no more than 12,000 g (4°C).
• The aqueous phase was transferred to other tubes. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains only in the aqueous phase. The volume of the aqueous phase is about 70% of the volume of TRIZOL LS Reagent used for homogenization.)
3. RNA Precipitation
• The RNA was precipitated from the aqueous phase by mixing with 3 microlitres of glycogen and 500 microlitres of isopropyl alcohol.
• The mixture was centrifuged for 30 minutes at 12,000 × g (2 to 8°C)
4. RNA Wash
1. The supernatant was removed. The RNA pellet was washed once with 75% ethanol, adding 900 microliters of 75% ethanol per 0.75 ml of TRIZOL LS Reagent used for the initial homogenization.
2. The sample was inverted and mixed and centrifuged at 12,000 rpm for 30 minutes at 4 degrees.
5. Redissolving RNA
• The RNA pellet was dried.
• RNA was dissolved in RNase-free water (or 0.5% SDS solution) by passing the solution through the pipette tip for a few times, and incubating for 10 minutes at 55 to 60°C.
Precautions for preventing RNase contamination:
RNases can be introduced into the RNA preparation at any point accidentally during the isolation procedure through improper technique. RNase activity is difficult to inhibit, so it is very essential to prevent its introduction. The following guidelines should be observed carefully while working with RNA. Always wear disposable gloves. Usually, our skin contains many bacteria and molds that can contaminate the RNA preparation and be a source of RNases. Good and clean microbiological techniques will prevent microbial contamination. Use sterile, disposable plastic ware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipment.
Determining RNA quality and quantity
RNA quality can be determined by examining the ratio of absorption at 260 nm and 280 nm with UV spectrophotometry. For high-quality RNA, A260/A280 ratio should be in the range of 1.9–2.1. RNA can be quantified by measuring the absorption at 260 nm, where 1 absorbance unit is equal to 40 μg/ml, at a pH of about 7.5. The validity of this ratio as a measurement of RNA purity was challenged. In addition, the quality of total RNA preparations should be examined through electrophoresis, where both 18S and 28S RNA bands should be very prominent.
RNA storage and stability
RNA solution from Ambion/Invitrogen and Qiagen are used for stabilizing cellular RNA in tissue samples, and for stabilizing final purified RNA. Ambient storage of tissue samples in RNAlater preserves RNA integrity similarly as storage at low temperature. RNA is based on the inhibition of RNAses by sulfate salts such as ammonium sulfate at specific pH. RNA quality can be checked using agarose gel electrophoresis. For long-term storage, RNA should be kept at -80°C, while the ethanol precipitate of RNA can be stored at -20°C.