Single‐Step Method of Total RNA Isolation by Guanidine–Phenol Extraction

Abstract

The original single‐step method is the first procedure to isolate purified total ribonucleic acid (RNA) from a variety of sources including tissues and cells from human, animal, plant, yeast, bacterial and viral origins, without the requirement of high‐speed ultracentrifugation. The method is based on liquid‐phase separation resulting in sequestration of pure RNA into the aqueous phase. RNA is precipitated from the aqueous phase, dissolved, reprecipitated and washed with alcohol before the final solubilisation step. The entire procedure can be completed in less than 4 h and it provides RNA that is suitable for many sensitive downstream applications such as RNase protection assays, northern blotting, sequencing studies and reverse transcription‐polymerase chain reaction. This pioneering methodology has served as the impetus for the development of newer and improved RNA extraction methodology that now enable investigators to extract and purify RNA in less than 60 min. Key Concepts: Enzymes within living cells rapidly degrade RNA after a tissue sample is removed from the donor. To prevent RNA degradation, tissue samples that cannot be immediately processed must be rapidly frozen with dry ice or liquid nitrogen and stored frozen at −80 °C until the RNA can be extracted. At the time of RNA extraction, frozen cells or tissues must be immersed in the denaturing solution and rapidly homogenised before the tissue thaws in order to inactivate RNAse and avoid RNA degradation. The quality of the recovered RNA is dependent on a delicate balance of salt concentration and optimal pH. Overloading the extraction solution with too much tissue or diluting the denaturing solution beyond what is specified in the protocol will impact the quality of the resulting RNA. The extraction of RNA from samples that have a high buffering capacity, such as blood, plasma or tissue culture medium, requires greater care in order to maintain optimal salt balance and pH control. Overdrying of the RNA pellets will impede RNA solubilisation. RNA should be solubilised at a concentration that will be appropriate for meaningful spectrophotometric quantitation as well as subsequent downstream molecular biology applications. Enzymes that are involved in RNA degradation are ubiquitous and special care must be taken to avoid RNAse contamination during RNA solubilisation and storage.

Genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in Croatia

“…Total RNA was extracted from 500 μL of NPSs using guanidinium isothiocyanate and phenol‐chloroform extraction . The cDNA was synthesized using random hexamers and MuLV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA) and the synthesis was carried out at 42°C for 60 min, followed by 95°C for 5 min.…”