In this blog, we have discussed an optimized method on the basis of a combination of two commercial kits for the extraction of soil nucleic acids. DNA and RNA are simultaneously extracted and purified ensuring no usage of harmful solvents. This process can be helpful to monitor the variations that may occur in DNA and RNA extraction from different types of soil. The process included a series of quantitative procedures by testing the quantity of starting soil, parameters of grinding, and the ending elution volume with DNA and RNA extraction Kit. This is helpful to ensure either of the kits do not saturate.

• To assess the fungal parameters, a soil-crushing step in liquid nitrogen is incorporated
• This procedure proved to be productive on various tropical soils such as Andosol. On the other hand, the greater content of clay-like poorly crystalline clay, Iron, and Aluminum oxides caused difficulties in nucleic acid extraction.
• The soil clay content was positively impacted by the correlation of earlier tropical soils with soil respiration, as compared to DNA

 

Method Details   A commercial DNA extraction kit for the safe and rapid recovery of DNA and RNA simultaneously was implied without making the use of any hazardous solvents. The DNA kit is based on an SDS extraction buffer which was previously proven to be efficient. RNA recovery was made possible because extra measures were taken to work under RNase-free conditions. Diethyl pyrocarbonate (DEPC) was used to treat all solutions and glassware to make sure that they are free of RNase. The usage of certified RNase- and DNase-free plastic tubes was ensured. The centrifugation was performed at a temperature as low as 4°C. The tubes were placed on ice until the next step. This resulted in purified RNA from the DNA washing solution. (Tournier, et al., 2015)

 

1- Soil Sample Preparation

 

A soil sample (250 mg) is added to a Lysing Matrix E tube and frozen at -80°C overnight. The testing of 125, 250, 500, and 750 mg samples is done and 500 mg samples resulted in the maximum of DNA recovery. 

 

2- Cells Lysing

 

978 μL sodium phosphate buffer and 122μL MT buffer is added. 20 mg caseine is added to the lysing mix for the extraction of nucleic acids from andosol. This is done for the saturation of the high adsorption limit of non-crystallized clays.

 

3- Grinding

 

The sample is homogenized in the FastPrep™ instrument (MP Biomedical) at a speed of 6.0 for 40 seconds. Then, the sample is cooled in ice for 5 minutes and moved on to the next grinding step under the same conditions. Initially, there was a single homogenizing step but a second grinding step was introduced to increase the recovered amount of nucleic acid for further analysis.  

 

4- Centrifuge

 

Centrifuge is done at 14,000 x g at 4°C for 5 minutes. The supernatant is transferred to a clean microcentrifuge tube.

 

5- Protein Precipitation

 

Incorporate 250μL protein precipitation solution. Mix the components by shaking the tube 10 times. Place it on ice until the next step.

 

6- Centrifuge again using the same parameters to pellet precipitate. Transfer the supernatant to a clean 2 mL microcentrifuge tube.

 

7- DNA Binding

 

The Binding Matrix suspension is suspended and 1.0 mL of it is added to the supernatant. It is then placed on a rotary shaker for 12 minutes at 23 RPM at room temperature. The allows the DNA to bind. Increasing DNA binding time from 5 to 12 minutes is useful to avoid DNA contamination into the washing solution which contains RNA. IT is centrifuged again using the same parameters but for 2 minutes to pellet DNA binding matrix. The supernatant is transferred to a clean tube to purify the RNA further.

 

DNA Purification Process

 

1- DNA Binding Matrix is re-suspended in a 500 μL of guanidine thiocyanate (5.5M). Transfer it to a SPIN™   Filter column and centrifuge again for a minute. Remove the eluate from the catch tube and transfer it to the RNA tube.

 

2- 500 μL prepared SEWS-M is added and the pellet is re-suspend using liquid force from a pipet tip.

 

3- Centrifuge with the same parameters for a minute. Empty the tube and replace it. 

 

The centrifuge and suspension processes are repeated until desired results are obtained. 

 

In the future, technical optimization has the potential to include recovery of RNA and DNA testing to calculate functional genes as compared to their presence in DNA, along with the recovery of enzymes from the residues obtained.

 

References

Tournier, Amenc, Pablo, Legname, Blanchart, Plassard, . . . Bernard. (2015). Modification of a commercial DNA extraction kit for safe and rapid recovery of DNA and RNA simultaneously from soil, without the use of harmful solvents. Elsevier, 2, 182-191.