Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Bradley Schmitz
Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies deoxyribonucleic acids (DNA), RT-PCR enables the amplification of ribonucleic acids (RNA) through the formation of complementary DNA (cDNA). This enables RNA-based organisms found within the environment to be analyzed utilizing methods and technologies that are designed for DNA.
Many viruses found in the environment use RNA as their genetic material. Several RNA-based viral pathogens, such as Norovirus, and indicator organisms, such as pepper mild mottle virus (PMMoV), do not have culture-based detection methods for quantification. In order to detect for the presence of these RNA viruses in environmental samples from soil, water, agriculture, etc., molecular assays rely on RT-PCR to convert RNA into DNA. Without RT-PCR, microbiologists would not be able to assay and research numerous RNA-based viruses that pose risks to human and environmental health.
RT-PCR can also be employed as a tool to measure microbial activity in the environment. Messenger RNA (mRNA) is the single-stranded template for protein translation, and measuring the levels of different mRNAs indicates which genes from which microbes are being expressed within the environment. Analyzing gene expression gives clues to what biological pathways are used by organisms to survive in different environmental conditions. In some cases, gene expression can be utilized to determine which organisms may survive best in harsh conditions and have capabilities for bioremediation of contaminated soil or water.
PCR is based on the amplification of DNA templates, which limits its use in detecting RNA from organisms. However, RT-PCR provides a means for using RNA to produce cDNA using specialized enzymes, known as reverse transcriptase (RT). This cDNA can then be used as the starting template for subsequent amplification with conventional PCR (Figure 1).
Reverse transcription can be controlled to amplify only desired products or an entire community of nucleic acids found within an environmental sample, depending on the primers that are used to template the cDNA synthesis. This is important, as soil and water samples are often saturated with various nucleic acids that aren’t desired for specific analyses. Random primers, which can bind to RNA sequences found in any type of microbes, can be used in RT-PCR to detect most RNA, so the sample can be analyzed for the presence and relative abundance of multiple organisms in the environment. On the other hand, sequence-specific primers initiate cDNA synthesis for precise sequences found in only one or a few organisms. This allows an environmental sample to be tested for a specific purpose, such as determining whether Norovirus, which can cause gastrointestinal illnesses in human, is present in water.
Figure 1. Step-by-step process of RT-PCR analysis of environmental RNA samples.
1. Sample Collection: Soil Sample
- Find a sample location via GPS, coordinates, or sight.
- For random sampling, choose random points within an area to get a general census of microbial habitats. Transect sampling collects from points along a straight line, e.g., adjacent to a streambed. Grid samples are systematically taken from points at regular intervals, and are useful for mapping microbial communities in an unknown or variable area.
- Sampling within a 3-6 inch (7.5-15 cm) depth provides access to the most abundant microbial activity that is near, but not within, the rhizosphere (the narrow region of soil directly affected by plant roots and their associated microorganisms).
- To collect the soil sample, push and twist a hand auger into the ground to the predetermined depth.
- Lift the auger. The soil is found within the hollow stem of the auger.
- Scrape the soil at the bottom of the auger into a soil collection bag. Be sure not to touch or contaminate the soil.
- Label the bag properly with location, name, date, and time.
- At the laboratory, pass the soil through a 2-mm sieve to remove any gravel and rock.
- Analyze a portion of the soil for soil moisture content. For details of this step, please refer to JoVE Science Education video on soil moisture.
2. Sample Collection: Water Sample
- Find the sample location via GPS, coordinates, or sight.
- Collect the water in a storage bottle. Record the volume of water collected; if microbes in the sample are quantitatively assayed, then the microbial concentration can be determined based on the collected volume.
- Immediately test the water for any parameters required for the experiment (temperature, pH, conductivity, salinity, nitrogen and phosphorous content, etc.) using the appropriate electronic probes.
- Place the bottle containing the water sample into a cooler with ice. Transfer the cooler to the laboratory.
3. RNA Extraction
- To collect and concentrate microorganisms from the environmental sample, please refer to the JoVE Science Education video on community nucleic acid extraction.
- Extract RNA from viruses using a commercial extraction kit according to manufacturer’s instructions.
- Briefly, first mix the samples with lysis buffer supplemented with ethanol, then add the samples into spin columns.
- Centrifuge the columns at approximately 12,000 x g, discard flowthrough. Add wash buffer to the columns and centrifuge again.
- Add RNase-free water to the columns and centrifuge for 30 s to elute RNA into sterile 1.5-mL low-adhesion microfuge tubes.
4. Reverse Transcription - PCR
- Retrieve the following reagents from the -20 °C freezer: dNTP, concentrated (e.g., 10x) reverse transcription buffer, primers (in this example, random primers). Thaw these on ice or at room temperature inside a clean hood, and keep them on ice once thawed. Also retrieve the reverse transcriptase and RNase inhibitor and keep them on ice.
- Calculate the reagent volumes needed to make a “master mix” that combines all the reagents constant among every reaction (see Table 1 for a sample reaction). Prepare enough master mix for every sample, as well as a positive control (using a known transcript template) and negative control (e.g., without reverse transcriptase, with only water as template, etc.) reactions. Include an extra 10% in volume.
- Assemble the master mix in 1.5-mL low-adhesion microfuge tubes. This minimizes the binding of reagent molecules to the tubes’ plastic walls.
- When reagents are thawed, add calculated volumes to the microfuge tube. Gently vortex and centrifuge each tube before addition. Make sure to change pipette tips between adding each reagent to prevent contamination.
- After all reagents have been added, vortex and centrifuge the master mix to ensure a homogeneous mixture. Put reagents back into storage at -20 °C.
- Prepare and label 8-tube PCR strips, designating one tube for each sample or control reaction.
- Aliquot an equal volume of master mix into each tube. Then, add reaction-specific components, such as the RNA extracts.
- Place the strip cap securely onto the PCR strip, and centrifuge in a minicentrifuge with a strip tube adaptor to ensure all liquid is collected at the bottom of each tube (Figure 2).
- Place PCR strip securely in thermocycler. Press down to ensure tubes are secured.
- Set the thermocycler to run the program appropriate for the RT being used (see Table 2 for a sample protocol). When the program is complete, the tubes will contain cDNA products, which can then be subjected to PCR amplification. For details, please refer to the JoVE Science Education videos on PCR and quantitative PCR. Store cDNA at -20 °C until use.
Figure 2. Capped 8-tube strip containing master mix and extract.
|Reagent||Volume per 1 reaction (μL)|
|10x RT Buffer||2.0|
|10x Random Primer||2.0|
|Molecular Grade H2O||3.2|
Table 1. RT Master Mix Ingredients.
|Step 1||Step 2||Step 3||Step 4|
|25 °C , 10 min||37 °C , 120 min||85 °C , 5 min||4 °C , ∞|
Table 2. RT Reaction Thermocycler Program.
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!
When RT- PCR is complete, some of the PCR product can be separated and visualized on an agarose gel (Figure 3). In this example, a gene-specific primer was used to detect for the presence of an RNA virus. Bands of the expected size are obtained from two of the samples and the positive control reaction, but not from the negative control, indicating the presence of this virus in two of the water samples being tested.
Figure 3. Gel electrophoresis of RT-PCR products. M: DNA size marker; P: positive control; N: negative control. Reactions using RNA from four water samples were run in lanes 1, 2, 3, and 5.
Applications and Summary
RT-PCR is necessary for creating cDNA from an RNA template. This enables RNA-based microorganisms to be analyzed utilizing molecular assays developed for DNA. Once the cDNA is synthesized, PCR assays can determine the presence or absence of RNA-based microorganisms within an environmental sample. This enables further downstream analysis to determine microbial ecology, health risks, and environmental risks.
RT-PCR can also be utilized to assay mRNA as a means to observe which genes are being expressed in an environment. This provides information about which proteins and pathways microbes rely on to survive in particular environmental conditions. Gene expression analyses can identify microbial pathways that breakdown environmental contaminants such as hydrocarbons or chlorinated solvents, and microbes with these pathways can be harnessed for bioremediation.
Risk assessment incorporates RT-PCR in order to analyze human and environmental health risks. Combining RT with quantitative PCR allows RNA viruses to be enumerated within samples, so that human and environmental exposure can be calculated for the purpose of Quantitative Microbial Risk Assessment (QMRA).