Extraction and isolation of viral RNA is the first step in the detection and testing of viral RNA by real-time RT-PCR. Here we compare different methods and protocols for extraction of viral RNA.
Nested RT-PCR and quantitative real-time PCR (qRT-PCR)-based detection of virus RNA are the preferred methods for the diagnosis of viral diseases (see Robert Koch Institute >). In contrast to examinations with ELISA or IFA, advances in nested and non-nested qRT-PCR-based screening means that patients can now be diagnosed much earlier in the infective process, and with greater precision and accuracy.
For the detection of viral RNA, it must first be isolated in order to enable subsequent analysis by real-time RT-PCR. There are several common methods: RNA extraction with purification kits and columns, the single-step method according to Chomczynski and Sacchi or the classical phenol-chloroform method. A problem common to all methods, apart from the toxic components, is the residual content of genomic DNA after RNA extraction. This contamination is of importance for downstream applications such as RT-PCR, since DNA still present can lead to false positive results. The DNA can be eliminated e.g. by DNase I > digestion or phenol-chloroform extraction. A degradation of the RNA can be prevented with an RNase inhibitor >.
The following protocols are designed to isolate and purify virus RNA. You will find an overview of the methods presented here in our online catalogue under RNA Tools >.
1. Extraction of virus RNA using commercially available cleaning kits
The principle of the RNA purification kits > ("spin column-based nucleic acid purification") is based on a lysis buffer for processing the cells and applying the homogenate to special cleaning columns. Chaotropic salts in the lysis buffer inactivate existing RNases.
Using Genaxxon’s Viral RNA and DNA Purification Kit > with special silica gel membrane technology, purified virus RNA is isolated within 30 minutes. The kit is suitable for isolation of RNA from plasma, serum, urine, cell culture supernatant, cell-free fluid or virus-infected tissue such as nasal or throat swabs.
The protocol is available here >.
The Total RNA Purification Mini Spin Kit > from Genaxxon allows the extraction of high quality RNA from tissue and cells. This kit is also suitable for isolation from RNA. The kit uses columns with membranes that bind nucleic acids efficiently and selectively at high concentrations of chaotropic salts. Any RNases are inactivated with guanidine thiocyanate and β-mercaptoethanol. The kit contains a novel Antifoam Reagent > that prevents foaming.
The Total RNA Purification Mini Spin Kit PLUS > also contains additional homogenization tubes with ceramic beads for the disruption of cells. The ceramic beads > are also available separately.
Purification in 96-well plates with membranes - High throughput screening
The 96-well Plate Total RNA Purification Kit > from Genaxxon is optimally suited for high-throughput screening. The kit uses 96-well plates with membranes that bind nucleic acids very efficiently and selectively in the presence of high concentrations of chaotropic salts. The homogenate is lysed with guanidine thiocyanate and detergents. Any RNases are inactivated by guanidine thiocyanate and β-mercaptoethanol. By adding ethanol RNA is bound on the membrane of the 96-well plate.
Genaxxon offers RNA Mini Spin Purification Columns > for RNA purification kits of other manufacturers.
2. RT-PCR directly from whole cells without RNA purification or cell lysis
The Genaxxon HotScriptase RT Cell Mastermix > allows RT-PCR directly from whole cells (link) without time-consuming and expensive RNA purification or cell lysis. The amplification is performed directly from the cells. The Cell RT Mastermix with HotScriptase RT Polymerase > allows RT-PCR directly from the cell suspension without an isothermal reverse transcriptase intermediate step. For example, RNA virus infections can be detected directly from various body fluids such as blood plasma, saliva or semen using RT-PCR without prior isolation or purification of RNA.
Genaxxon´s HotScriptase Covid-19 master mix > was successfully used by several customers for the detection of corona viruses (SARS-CoV-2, COVID-19).
3. Extraction of RNA using the single-step method (according to Chomczynski and Sacchi >), as an extension of the two-phase method
GENAzol > contains guanidinium thiocyanate and phenol. Guanidinium thiocyanate has a denaturing effect, i.e., it lyses cells and inhibits RNases. Proteins and DNA that are present in the sample, are dissolved in the organic phenol. Chloroform is added to the sample and is homogenized with GENAzol. The homogenate then separates into a clear aqueous top layer (containing RNA), a boundary layer and an organic bottom layer (with DNA and proteins). RNA is precipitated from the aqueous layer with isopropanol. The precipitated RNA is washed to remove contaminants and then resuspended for use in subsequent applications.
4. Self-made cell lysis and extraction buffer solutions
Due to supply shortages of commercially available RNA isolation kits and their components, this can be a real alternative for laboratories. The use of the solutions with columns of conventional cleaning kits led to comparable detections in the subsequent qRT-PCR.
Senior Investigators, Stefanie Deinhardt-Emmer and Ulrich S. Schubert of the Friedrich Schiller University, Jena >, recently tested the effectiveness of six easy-to-manufacture buffer solutions that could be used to extract virus RNA from respiratory material from positively tested SARS-CoV-2 patients (publication in press).
The buffers that generated the highest yields of intact virus RNA, based on their qRT-PCR Cq value were guanidinium thiocyanate buffer solution with 2-mercaptoethanol and guanidinium thiocyanate without 2-mercaptoethanol.
a. Buffer solutions based on the denaturing and chaotrophic substance guanidinium thiocyanate
- Buffer solution with 2-Mercaptoethanol: Due to the inherent toxicity of 2-mercaptoethanol for humans and for water, the use of protective clothing and working under a fume hood is required.
Buffer solution without 2-mercaptoethanol: The toxic 2-mercaptoethanol is not included here. However, the buffer solution without 2-mercaptoethanol has a slightly lower Cq than that with 2-mercaptoethanol according to the study led by Deinhardt-Emmer, & Schubert et al (in press).
According to the Federal Environment Agency, the octylphenol derivative Triton X-100 is classified as a particularly worrying substance due to its endocrine effect on the environment.
b. Guanidinium thiocyanate-free buffer solutions
These are less efficient. If the potent guanidinium salt is not available, these buffer solutions can be used.
- Buffer solution with Triton X: According to the Federal Environment Agency, Triton X-100 is classified as a substance of very high concern because of its endocrine effects on the environment.
- SDS lysis buffer
The Charité in Berlin, a designated reference laboratory for SARS-CoV-2 virus screening, has published a pipeline for the diagnostic detection of SARS-CoV-2 by qRT-PCR:
https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2)
