Our last three soil articles were focused on how to get DNA from soil. In great detail we covered the topics of homogenization and the chemistry (and specifically the lysis solutions) needed to isolate high yields and purity DNA.   Working with DNA is far less stressful compared to working with RNA.  Yields are always higher and there is no worry about degradation.

RNA however…

RNA Isolation from soil is one of the most difficult applications we perform in environmental molecular biology. RNA purification is always an arduous task and from soil it becomes a bigger challenge.  One of the biggest problems is the yield of RNA from soil. Because typical yields of RNA are so much lower than for DNA, usually between 10-20% of the yield of DNA, starting with a larger amount of sample is desired. This requires a method that uses larger tubes (15 ml) and a bigger centrifuge.

The other major issue is, of course, the humic acid and inhibitor content of soil co-contaminating the RNA.  Purity for RNA applications is even more important because dilution of the RNA for reverse transcription is not desired when looking for low copy genes. The RNA needs to be concentrated when added to the reaction and inhibitors cannot be present.

Isolation of RNA from soil has special requirements

For these reasons, MO BIO developed a completely different process to purify RNA from soil that does not use silica spin filters. Today we are going to talk about the RNA PowerSoil Kit  and how to achieve the best possible results.

The protocol is a combination of methods. It uses IRT for inhibitor removal, phenol-chloroform extraction for complete microbial lysis, and anion-exchange for high quality purification.  The end result is the isolation of very clean RNA in the volume desired allowing for maximal use in RT-PCR.

Today I would like to share with you some tips and tricks for using this method to minimize the amount of troubleshooting or optimization you need to do. Because every soil is different in texture, moisture, and microbial load, soils can behave differently during extraction.   Let’s go through the protocol and discuss the key steps where problems may occur and where changes can be made to improve the results. Let’s start at step 1…..

1. Starting sample (step 1): For most soils, 2 grams of soil should be the maximum amount used. However, for sediments, the wet weight results in much less actual soil in the prep and reduced yields from samples that already have low microbial load. With sediment samples, I have used up to 5 grams wet weight of sample. If there is significant water sitting on top of the soil, you can centrifuge the sample briefly after adding it to the bead tube and remove the excess water.

2. Phenol: Chloroform: Isoamyl alcohol (PCI) type (step 5):  It is important to use the correct PCI and we give some recommendations in the manual. The phenol should be a 25:24:1 ratio of PCI and the pH should be between 6.7 and 8 and stored under TE buffer pH 8.0.   Many people want to use an acidic phenol for the prep because low pH phenol is sometimes used for RNA preps for other samples such as animal tissues and cells. For soil, we do not recommend this.  Stick with the neutral pH phenol for best results.

3. Isopropanol precipitation optimization (step 12): After PCI extraction and the addition of Solution SR3, the next step is an isopropanol precipitation to isolate the total nucleic acids. If you started with sediments, you may have more than 5 ml of sample after adding SR3. Increase the amount of SR4 (isopropanol) to equal the volume of sample at step 11 to ensure a complete precipitation.

4. Isopropanol precipitation temperature (step 12): The standard protocol recommends freezing the samples at -20^oC. For samples with high salinity perform the precipitation at room temperature. The freezing temperature will cause the salt to precipitate and change the binding conditions to the anion-exchange column in the purification.  You will know if the sample precipitated salt by the way the pellet looks. It should be flat and glossy, like a normal RNA pellet. If it is large and crusty, you have some salt in there.   Sediment samples, because of the excess water, tend to be salty, even from freshwater lakes. 

Stopping Point: I have extended the incubation at step 12 for longer than 30 minutes and even overnight and the RNA was fine. I wouldn’t recommend it for every sample and you may want to test it for your soils.  In an emergency, you can delay or stop here.

5. Anion-exchange column flow issues (step 15):  The columns used for the final purification of the RNA are a packed resin that flows using gravity to drip through the column.  Sometimes these can move slowly because of packing down of the resin. To help increase the flow of the buffers and sample through the column, we will sometimes use positive pressure to gently motivate the buffers through the resin.  If the column is still having difficulty with the flow rate, we will use the syringe and barrel from a 5 ml syringe to apply light pressure to the column to enhance the flow. To do this, hold the barrel of the syringe flush against the opening of the column. Push the syringe plunger through the syringe, holding the barrel so that the air does not escape around the top of the column. Very gently apply the pressure. Do not exceed a flow rate of 1 drop per second.

6. Shake, shake, shake Solutions SR5 and SR6 (step 15): Give your solutions SR5 and SR6 a good shake before use to ensure the components are well mixed. Sometimes solutions containing isopropanol can separate while sitting on the shelf and are not homogenous unless mixed first. A few good shakes will do the trick.   

7. Elution time-saving tip (step 19-20): I sometimes elute directly into my 2 ml collection tube instead of into the 15 ml tube to save a transfer step and some plastic. Make sure the gravity column is balanced on the collection tube in a rack in a way that it can’t fall over. This tip is for the technically savvy. Don’t try this if you are using the kit for the first time.

8. Final isopropanol precipitation (step 20): After elution from the gravity flow column, the final precipitation is done using the isopropanol again (Solution SR4).  This is incubated at -20^oC. Do perform this step at -20^oC (vs. room temperature). Extended time at this step is ok.

Stopping point: If you can’t finish the prep, this is an ok place to stop for the night. The sample is frozen at -20^oC and the RNA is stable.

9. The RNA pellet (step 22): After centrifugation to collect the RNA from the isopropanol, the normal pellet will be small and glassy. Make sure to orient the tubes in the centrifuge the same way so you can quickly identify where the pellet is in all of the tubes when you decant the isopropanol. When drying the pellet, to make the process go faster, we like to place the tubes inverted onto a kemwipe placed on the air flow intake of the tissue culture hood while it’s on.

10. RNA resuspension (step 23): Now that you have a nice dry pellet, resuspend the RNA in a volume based on what you need for reverse transcription. In our lab, if the soil has a high yield of microbes, we’ll resuspend in 50-100 of water (usually the final concentration is ~100-200 ng/ul). For sediments and dry soils with low microbial biomass, we’ll use 25 ul so the RNA is more concentrated for use. This step is flexible and you can use the amount of water to resuspend the pellet that is best for you.

Bonus Tip: You can isolate DNA from the column also since most of it stays behind after elution of the RNA in Solution SR6. To get the DNA out, we have Solution SR8 (from the DNA Elution Accessory Kit) that has a higher salt concentration and will elute the genomic DNA. And since the isolation procedure is very gentle, the DNA molecular weight is very high.  An additional benefit of the anion-exchange column method is the ability to get the RNA and DNA from the same sample and eluted in two different tubes.

Double Bonus Tip: RNA Stabilization and Storage in Soil:

We are often asked about the stabilization of RNA in soils upon collection and the use of RNALater for soils.  RNALater is not compatible with soil. We have performed time-courses of soil stored in RNALater at various temperatures and found that RNALater results in excessive humic acid release and co-purification with the RNA that cannot be removed with anion-exchange.  The longer the storage, the darker the sample becomes. 

To help those researchers that need to stabilize soils upon collection and want to ensure that the microbial profile remains constant during transport, we use LifeGuard Soil Preservation Solution. The composition of this solution results in stasis of the microbial content and isolation of intact RNA regardless of the length of time in storage or temperature.  The ratio of LifeGuard to soil can vary based on the content and the microbial load (wet soils and sediments should use more and for normal soils we use 2.5 ml per gram of soil).   More information including data can be found here.

Summary:

The most challenging sample extraction is RNA from soil. No other extraction procedure requires isolation of highly labile low abundance RNA in the presence of so many inhibitors and microbial RNases.  But, high yields of clean RNA are possible.  I hope these ten tips will save you some time when you have a new soil sample that you want to extract.  If you have some of your own short cuts, tips and tricks, and advice, let us know. We love to hear how researchers make changes to get the results they need.

Thanks for reading our blog and we always welcome comments and feedback. You can leave it here or email us at technical@mobio.com.

Warm wishes from Analytica in Munich,

~Suzanne