In the previous article we introduced how to extract soil DNA. A large section explains in detail the issues of grinding homogenization and chemical reagents (with a focus on lysis buffer) that affect DNA yield and purity. DNA is far from doing RNA stress. Don't worry too much about degradation, the yield is quite high.

RNA is different. . .

Extracting soil RNA is one of the most difficult research methods in environmental molecular biology research. RNA extraction itself is a daunting task, and RNA extraction from soil is more challenging. One of the biggest difficulties is the soil RNA yield. RNA is much less abundant than DNA from the soil. The former is only 10-20% of the latter, so it requires more loading than DNA. Therefore, a larger grinding tube (15 ml) and a larger centrifuge are required.

Another big problem is the contamination of inhibitors such as humic acid. RNA experiments require high purity, and when reverse transcription is sought for low copy number genes, you cannot dilute RNA samples. The concentration of RNA required to be added to the reaction system is sufficiently high and cannot contain inhibitory factors.

Special conditions are required to extract soil RNA

Therefore, MO BIO has developed a method for extracting soil RNA without using a silica spin column. The following discussion about the RNA PowerSoil Kit and how to get better extraction results.

This set of operations involves a variety of methods. The IRT technique removes the inhibitory factor, and the phenol chloroform method most fully lyses the microbial cells, and the anion exchange technique has a higher purification quality. Ultimately, very clean RNA is obtained to maximize the need for RT-PCR.

Let's discuss how to use these techniques and rework as little as possible. The texture, moisture content and microbial abundance of each soil sample are different, and the conditions appearing during the extraction process are not the same. Below we focus on a few key steps that are error-prone and what changes can be made to get a better extraction.

1. Sample start amount

For most types of soil, 2 g of soil is the largest sample start. However, for soils with higher water content such as sediment, the actual soil content is lower and the microbial content is not high. I use up to 5g of starting amount. If the sample is added to the bead casing, it is found to have a layer of water on it. It is best to centrifuge and remove excess water.

2. Phenol: Chloroform (step 3):

It is very important to use the correct PCI, and we give some suggestions for use in the kit operating instructions. The PCI ratio must be 25:24:1, pH between 6.7-8, and stored in TE buffer at pH 8.0. Many people extract animal tissues and cells habitually using only low pH phenol to extract RNA. For soils, pH neutral phenol will be suitable.

3. Optimize isopropyl alcohol concentration (step 9):

After the PCI lysis step, the SR3 solution was added, and the next step was to add isopropanol to precipitate the total nucleic acid. If you are doing sediment, the total volume may exceed 5ml after adding SR3 solution. The amount of SR4 (isopropyl alcohol) used was increased to the same volume as that obtained in step 8, to ensure sufficient precipitation of the nucleic acid.

4. Ambient temperature of isopropanol precipitation (step 9):

The original operating instructions recommend freezing the sample at -20 °C. The sample nucleic acid precipitation step with high salt concentration should be carried out at room temperature. The salt precipitated from the frozen sample changes the nucleic acid adsorption binding conditions on the anion exchange spin column. You can see from the sediment whether there is salt precipitation. The normal RNA precipitation surface is flat and shiny, and if the precipitation is significantly larger and the surface is shelled, it indicates salt deposition. The sediment sample has a large water content, even if it is a freshwater lake. The excess water will contain salt. If your soil type is incubated at -20 °C to get a better yield, please follow the original method.

Pause point: I have extended the incubation time of step 9 for more than 30 minutes, even overnight, and the final RNA is still intact. I don't recommend extending the incubation time for each sample. At least your soil sample needs to be tested to know if it affects RNA quality. In special cases, you can pause at this step slightly.

5. Anion exchange spin column solution is naturally dripped (step 14)

The final RNA recovery step uses a spin column packed with resin and the solution is passed through a spin column using gravity. Sometimes the speed at which the solution passes through the resin is very slow, and a positive pressure can be appropriately applied to accelerate the flow. Usually our practice is to use a 5ml syringe needle to slowly pressurize, but the flow rate should not exceed 1 drop per second.

6. Shake, shake, and shake SR5 and SR6 (step 12):

SR5 and SR6 must be shaken well before use. Some solutions containing isopropyl alcohol will stratify when left to stand, just shake well before use.

7. Tips for saving elution time (steps 16-20):

Sometimes I will elute directly into the 2ml Collection Tube instead of the 15ml Tube. Make sure the spin column is placed vertically without tipping over. Only if you have mastered the skills, you can do so, and you are not allowed to do it.

8. Final isopropanol precipitation (step 17): Final isopropanol precipitation (step 20):

After elution from the centrifuge tube, isopropanol (SR4 solution) was added for final precipitation. Need to incubate at -20 °C. This step must be incubated at -20 ° C (VS room temperature). This step can be extended by the appropriate time.

Pause point: If the extraction work can't continue, you can pause overnight at this step. The sample is frozen at -20 ° C and the RNA can be stored stably.

9. Precipitate RNA (step 18):

Isolation of RNA by centrifugation of isopropanol, normal RNA precipitation should be small and glassy. All tube tubes should be oriented towards each other during centrifugation to facilitate rapid identification of RNA pellets when pouring isopropanol. Drying the RNA precipitation step needs to be done quickly. Usually our practice is to invert the Tube tube on absorbent paper in a clean bench.

10. Resuspend the RNA (step 20):

Now you have a beautiful dry precipitate. Resuspend RNA according to the volume requirements of reverse transcription. In our laboratory, if the sample soil is very rich in microbes, we will resuspend with 50-100 μl of water (usually a final concentration of 100-20 ng/μl). The sediment or dry soil has low microbial content. To increase the final RNA concentration, we resuspended in 25 μl of water. This step is very operability, please add the appropriate amount of water to resuspend the sediment according to your needs.

Additional tips:

After eluting the RNA with the SR6 solution, the DNA was centrifuged through the column filter. You can elute the genomic DNA with a SR8 solution (DNA Elution Accessory Kit component) containing a high concentration of salt. Because the entire nucleic acid extraction process is relatively mild, you can get larger molecular weight DNA. Another advantage of anion exchange spin columns is that you can extract RNA and DNA from the same sample.

Another additional tip: RNA stability and soil sample storage:

We are often asked about the stability of RNA in samples after sampling and the use of RNALater. In fact RNALater is not compatible with soil. We have performed RNALater protection experiments on soil RNA at different temperatures and times. It was found that with the prolongation of storage time, RNALater will release more humic acid from the soil sample, and adhere to RNA to adsorb with RNA. It is difficult to remove on the anion exchange spin column filter. The longer the save time, the darker the sample color becomes.

We use the LifeGuard Soil Preservation Solution to protect soil RNA during sampling and to ensure stable microbial structure during transport.

to sum up:

The most challenging sample extraction is soil RNA. The RNA in the sample is very unstable and low in content, and there are a large number of inhibitors and microbial source RNase. But getting high yield clean RNA can still be done. I hope that the above ten tips can shorten the process of extracting new soil-like RNA. If you have more extractions, let us know. We are happy to hear researchers get the results they want through certain steps and specific modifications.

Product Link: RNA PowerSoil® Total RNA Isolation Kit