to BioTechniques free email alert service to receive content updates.

Isolation of DNA and RNA from soil using two different methods optimized with Inhibitor Removal Technology® (IRT)
Suzanne Kennedy, Ph.D., Director of R&D
Sponsored,vendor-submitted protocol    Sponsored by Mo Bio Laboratories, Inc.    Published in November 2009 (p.19) DOI: 10.2144/000113290

Abstract

Environmental researchers face the difficult task of studying microbial communities from remote locations and under extreme conditions. Two major issues facing soil microbiologists are the preservation of the original microbial population during transport and storage and the removal of enzyme inhibitors from DNA and RNA to ensure successful gene expression or genotyping analysis. Methods for the stabilization of soil samples during transport and options for the isolation of RNA or DNA from environmental samples are described.

Introduction

Figure 1. In collaboration with Dr. Charles Lee (University of Waikato, New Zealand) the efficacy of LifeGuard™ was demonstrated by microbial community analysis of Antarctic Dry Valley soils collected on site. All samples were shipped at 4°C, either stabilized with LifeGuard™ or unstabilized, and stored for 30 days. RNA was extracted using the RNA PowerSoil® Total RNA Isolation Kit, and T-RFLP analysis was performed for 16 s rRNA. Results demonstrated that sample A soil stabilized and unstabilized were 86% similar in the number of phylotypes. The unpreserved sample A had additional phylotypes, indicating that the fluctuations in temperature during transit resulted in changes to the original community metatranscriptome. Sample B was Dry Valley soil collected at a totally different site, and as expected, gave a completely different transcriptional profile.

Environmental samples are inherently problematic due to inhibitors that cause PCR failure and inaccuracy in metagenomic analysis from irreplaceable samples. Typical inhibitors include humic acids, fulvic acids, polysaccharides, and polyphenols. Using the patented Inhibitor Removal Technology® (IRT), removal of PCR inhibitors from the final DNA or RNA is accomplished in even the most difficult samples. An equally important concern involves preservation of the microbial metatranscriptome in samples at the time of collection. A novel bacteriostatic solution that freezes the microbial content of soils during transport and storage and protects the RNA from degradation is the LifeGuard™ Soil Preservation Solution. Using LifeGuard™, soils stored at 4°C were stabilized, and total RNA was isolated suitable for terminal restriction fragment length polymorphism (T-RFLP) analysis. The combination of LifeGuard™ with the RNA PowerSoil® Kit results in a complete system for obtaining high quality RNA and DNA from soil.

Materials and methods

test LifeGuard™ Soil Preservation Solution, 100 mL (Cat. no. 12868-100; MO BIO Laboratories)

RNA PowerSoil® Total RNA Isolation Kit, 25 preps (Cat. no. 12866-25; MO BIO Laboratories)

RNA PowerSoil® DNA Elution Accessory Kit, 25 preps (Cat. no. 12867-25; MO BIO Laboratories)

PowerSoil® DNA Isolation Kit, 50 preps (Cat. no. 12888-50; Mo Bio Laboratories); 100 preps (Cat. no. 12888-100; MO BIO Laboratories)

Results

Preservation of microbial communities in Antarctic Dry Valley soil using LifeGuard™ Soil Preservation Solution

test T-RFLP results from LifeGuard™ stabilized soil indicate the soil microbial community was preserved during transport from Antarctica and New Zealand (Figure 1).

Isolation of high yields and quality RNA and DNA from the same soil sample using the DNA Accessory Kit

In Figure 2, the lanes are respresentative of the following: lane 1: ladder; lane 2: mulch RNA; lane 3: mulch DNA; lane 4: strawberry field RNA; lane 5: strawberry field DNA; lane 6: compost RNA; lane 7: compost DNA; lane 8: ladder.

The PowerSoil® DNA Isolation Kit gives 100% Success

PCR amplification of genomic DNA isolated from eight different soils using the PowerSoil®DNA Isolation Kit and kits from two other suppliers (Figure 3).

Only the MO BIO kit gives 100% success.

Summary

For preservation of soils and isolation of DNA and RNA from environmental samples, MO BIO offers the widest selection of methods suitable for metagenomic and metatranscriptome analysis. Trust irreplaceable samples to the experts at MO BIO Labs.

More Information
 
Do you have question about this protocol? Would you like more information about this protocol?
Submit your questions here and Biotechniques will forward your question and contact information from our database to an expert at Mo Bio Laboratories, Inc. who will contact you.
Yes, please forward my question, along with my name, title, company, and e-mail address to Mo Bio Laboratories, Inc.
BioTechniques will forward your question to the company, who will contact you directly by email.
Request more information
 
Name Question
Job Title
Company
Email
Contact Information
Protocol Categories
rightcontent.jsp called otherwise condition rightcontent.jsp called 222222222222222222222
Search Protocols
Search our protocols for your specific technique.
Submit Your Protocols
BioTechniques is always looking for author-submitted protocols related to peer-reviewed papers, so submit your protocols today to our editorial team. Suppliers or advertisers interested in publishing their protocols should also submit.
Click here for more information