RNA Analysis of Environmental Samples Using RT-PCR

1. Sample Collection: Soil Sample

1. Find a sample location via GPS, coordinates, or sight.
2. 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.
3. 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).
4. To collect the soil sample, push and twist a hand auger into the ground to the predetermined depth.
5. Lift the auger. The soil is found within the hollow stem of the auger.
6. Scrape the soil at the bottom of the auger into a soil collection bag. Be sure not to touch or contaminate the soil.
7. Label the bag properly with location, name, date, and time.
8. At the laboratory, pass the soil through a 2-mm sieve to remove any gravel and rock.
9. 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

1. Find the sample location via GPS, coordinates, or sight.
2. 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.
3. 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.
4. Place the bottle containing the water sample into a cooler with ice. Transfer the cooler to the laboratory.

3. RNA Extraction

1. To collect and concentrate microorganisms from the environmental sample, please refer to the JoVE Science Education video on community nucleic acid extraction.
2. Extract RNA from viruses using a commercial extraction kit according to manufacturer’s instructions.
3. Briefly, first mix the samples with lysis buffer supplemented with ethanol, then add the samples into spin columns.
4. Centrifuge the columns at approximately 12,000 x g, discard flowthrough. Add wash buffer to the columns and centrifuge again.
5. 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

1. 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.
2. 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.
3. Assemble the master mix in 1.5-mL low-adhesion microfuge tubes. This minimizes the binding of reagent molecules to the tubes’ plastic walls.
4. 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.
5. 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.
6. Prepare and label 8-tube PCR strips, designating one tube for each sample or control reaction.
7. Aliquot an equal volume of master mix into each tube. Then, add reaction-specific components, such as the RNA extracts.
8. 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).
9. Place PCR strip securely in thermocycler. Press down to ensure tubes are secured.
10. 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
25x dNTPs	0.8
10x Random Primer	2.0
Multiscribe	1.0
Rnase Inhibitor	1.0
Molecular Grade H2O	3.2
Total Volume	10

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.