RT-PCR 法を用いた環境試料の RNA 解析

Reverse Transcription Polymerase Chain Reaction, or RT-PCR, enables the detection of RNA viruses and microbial gene expression in the environment.

Unlike cellular organisms from humans to bacteria, which use DNA as their genetic material, some viruses have genomes made of RNA, including many pathogenic viruses such as flu, Ebola, and HIV. While some viruses can be detected by observing the pathogenic phenotype that develops when used to infect cells in cell culture, a majority has no culture-based characterization methods. RT-PCR can be used to detect for the presence of these viruses in the environment with high sensitivity.

At the same time, organisms with DNA-based genomes “express” the genetic information encoded in their DNA genomes by “transcribing” them into messenger or “m”RNA, which can then be “translated” into proteins to perform enzymatic or structural function in cells. As microbes respond and adapt to changes in the environment by turning on or off various genetic pathways, RT-PCR can be used to monitor such alterations in gene expression to serve as potential ecosystem assessors.

This video will go over the principles behind RT-PCR, outline a generalized procedure to perform this technique, and lastly, examine some of the ways this method is being applied in environmental microbiology today.

There are two key parts to this procedure: the extraction of RNA from biological samples, followed by its reverse transcription into DNA.

The isolation of RNA involves lysing cells in the sample by adding a detergent that breaks down lipids and proteins, followed by mechanical disruption. Extracting RNA from viruses usually requires the addition of proteinases, which are protein-digesting enzymes, to break down the viruses’ capsids – the tough protein coats that form the viral particle. On the other hand, when obtaining RNA from cellular organisms, the lysate may need to be treated with DNA-degrading enzymes, or DNAases, to avoid downstream results being confounded by the presence of the chemically similar DNA.

RNA can then be purified from lysates in one of two ways. In one method, known as acid guanidinium thiocyanate-phenol-chloroform extraction, the lysate is mixed with an organic-aqueous mixture that is then centrifuged to separate the phases. Proteins will be partitioned into the organic phase, lysed cell debris into the cloudy interphase, and nucleic acids into the aqueous phase. The upper aqueous phase can then be collected, and RNA is precipitated by the addition of alcohol such as isopropanol, which decreases the nucleic acid’s solubility in water.

In column-based RNA isolation, the lysate is mixed with alcohol and then passed through a gel-filtration column with a negatively charged resin, such as silica. The lysate is prepared so that positively-charged ions in the buffer form “salt bridges” that allow the negatively charged phosphate backbone of nucleic acids to bind to the resin. All other contaminants are then washed away. The nucleic acids are “eluted” from the column using a low-salt buffer or water. Because single-stranded RNA has fewer negative charges than double-stranded DNA, it can be preferentially eluted using buffers of specific concentrations.

Once isolated, the RNA is transformed into the more chemically stable DNA. Scientists have harnessed an enzyme naturally encoded by retroviruses like HIV. This enzyme is known as reverse transcriptase, or “RT”.

RT synthesizes complementary or “c”DNA from a short stretch of nucleotides known as a primer. This primer can have different sequences, depending on the purpose of the experiment. For example, the primer can be designed to have a specific sequence, in order to detect specific genes or organisms. Once synthesized, this “first strand” cDNA can be amplified by regular PCR.

Now that we have an understanding of the principles behind RT-PCR, let’s look at a protocol for performing this technique on RNA samples collected from the environment.

To begin the procedure, find a sample collection location using GPS coordinates or sight. Choose random points within an area to get a general survey of microbial habitats.

To collect the solid sample, push and twist a hand auger into the ground soil to the predetermined depth. When the auger is lifted, soil can be found within the hollow stem of the auger.

This soil is scraped directly into a soil collection bag and the bag is labeled with the location, name, date, time of collection and any other necessary information.

Transfer the soil to the laboratory and pass the soil through a 2-mm sieve to remove gravel and rock. Analyze a sample of the soil for moisture content, which can be informative about the level of microbial activity in the soil. To do this, refer this collection’s video “Determination of Moisture Content of Soil”.

For water sample collection, choose a site of interest similar to soil collection, and collect water into a sterile thick-walled plastic bottle. Take note of the volume of water collected. Test the water immediately for parameters like temperature, pH, and salinity, which can provide important information about expected microbial levels in the sample. Then, place the bottle in the cooler and transfer to the laboratory.

Microorganisms such as viruses are collected and concentrated from the environmental sample.

RNA can be extracted from the collected viruses by the spin column method using commercial RNA extraction kits according to manufacturer’s instruction. Lysis buffer, supplemented with ethanol, is first mixed with the sample. Place the appropriate number of spin columns into collection tubes, then apply the samples onto the column matrix. Centrifuge the columns at approximately 12,000 x g for 1-2 min to let the nucleic acids bind to the column, and discard the liquid flow-through.

Add wash buffer to the columns and centrifuge again. Place the columns into new, sterile 1.5-mL low adhesion microfuge tubes. Then, add water that is free of RNases, which are enzymes that degrade RNA, to the columns and centrifuge for 30 s to elute the RNA. The eluted RNA can be stored at -80 °C until use.

Before the experiment, appropriate primers should be designed in order to detect the sequences of interest, which can serve as markers for the presence of specific microorganisms.

Take out the RT reagents stored at -20 °C, and thaw the frozen reagents on ice (or at room temperature). These include nucleotides, reverse transcription buffer, and primers. Once thawed, keep the reagents on ice; the reverse transcriptase enzyme, and RNase Inhibitor should always be kept on ice.

While the reagents are thawing, calculate the volumes and concentrations of the components of the reaction. Once the reagents are thawed, gently vortex and spin each tube to ensure the contents are well mixed.

Working in a laminar flow hood to avoid contamination, assemble the components in a master mix to add to each PCR tube. When assembling the master mix in an Eppendorf tube, make sure to change pipette tips between each reagent to prevent contamination. After all the reagents have been added to the Eppendorf tube, gently vortex and spin down the master mix tube to ensure a homogeneous mixture.

Label a set of PCR tubes, making sure to include controls. Aliquot an equal volume of the master mix into each tube. Then, add reaction-specific components, such as the RNA extracts or water for negative control.

Once all the components are added, place the tubes in a thermocycler and set the reverse transcription program to run. The cDNA can then be subjected to PCR amplification. For a detailed description of this step, refer to this collection’s video on PCR.

When the PCR is complete, some of the PCR product can be separated and visualized on an agarose gel.For example, a gene-specific primer was used here to detect for the presence of an RNA virus. Bands of the expected size are obtained from the RT-PCR reaction but not from the negative controls, indicating the presence of this virus in the water sample being tested.

The identification of RNA-based microorganisms by RT-PCR enables analysis of ecological health, environmental risks and the conservation of biodiversity.

The use of microorganisms to clean up hydrocarbons and solvents from polluted soil and water represents an ecosustainable remediation alternative. Understanding native microbial gene expression can highlight specific microbial pathways that break down contaminants under these conditions. RT-PCR is often utilized to amplify mRNA from such environmental samples.

Public health measures often require rapid surveillance of viral sources of infection in the environment. Avian Influenza, for example, is highly infectious and can quickly spread to poultry, livestock, and even humans. In this particular example, researchers looked for the threat of Avian Influenza spread by wild birds.

Using a portable RT-PCR setup and apparatus, they screened a range of wild birds, even in remote areas, to detect infections early and prevent transmission.

Finally, RT-PCR can be used to characterize “biopesticides” being developed to target pests such as the glassy-winged sharpshooter, Homalodisca vitripennis, the host for a disease that severely damages grapevines in North America. A novel single-stranded RNA-virus is being developed as an agent to infect this insect. Using a combination of Homalodisca cell culture and RT-PCR confirmation of viral load, these authors were able to propagate a high concentration of virus for use as a biological control agent.

You’ve just watched JoVE’s introduction to Analyzing RNA-Based Environmental Organisms by RT-PCR. You should now understand the theory behind the protocol, how to apply the technique to your research, and some of the ways in which it is being used in the field today. Thanks for watching!