Authors: Roey Angel
The following protocol is intended for the simultaneous extraction of DNA and RNA from various soil samples along with suggestions on how to tweak the protocol for soil with higher humic content. The protocol has been used by many and results in very high yields of nucleic acids, typically much more than commercial kits. For buffers and solutions used in this protocol, please see accompanying document Buffers and Solutions for TNA Extractions.pdf.
This protocol is a general procedure for the co-extraction of DNA and RNA from soils. The procedure results in very high yields of nucleic acids and is suitable for all downstream enzymatic reactions. The protocol has been published in: Angel et al. 2012
NA extraction considerations
RNA handling: as RNA is very sensitive to both chemical and enzymatic degradation meticulous measures of precaution must be taken. The use of DEPC and DEPC-treated water is not recommended since the substance is toxic and its application was found to be nearly unnecessary (see: http://www.ambion.com/techlib/tb/tb_178.html). Instead, I recommend to simply work with clean, preferably baked, glassware (3h at 180 °C), to clean surfaces and spatulas with RNase eliminating solutions (such as RNaseZap®), and to use fresh and clean reagents for the preparation of the different buffers and solutions. As a general rule, work with RNA should be quick, precise, and tubes should be kept on ice. In my experience, this is much more important than any attempt to eliminate all RNases from apparatus and solutions. One should also bear in mind that soil samples contain infinitely more RNases than any contamination which might exist in the buffers or the apparatus. The only exceptions to this are the PEG Precipitation Solution, the Low TE Buffer, and the RNA storage solution. Since these are used to store RNA for relatively long periods of incubation extra care must be taken during their preparation to ensure highest degree of purity. For the preparation of the PEG Precipitation Solution I simply weigh everything into a Schott Bottle and add approximately the right amount of water as I prefer to be somewhat inaccurate in the concentrations than to have to transfer the solution between vessels and risk contamination.
Selection of extraction buffer: The TNS buffer used in this protocol is based on The TNS extraction buffer is based on the one used by Henckel et al. 1999 TNS buffer results in higher RNA yields and more intact rRNA (more intact 23S band). However, TNS tends to carry much more humic substances into the nucleic acids phase compared to TNC buffer which could make it ineffective for soils with high organic matter content.
Use of phenol in the bead beating process: the use of phenol increases the yield up to 4 times compared with the phenol free option, but also increases the carryover of humic substances. If you choose not to add phenol simply double the amount of extraction buffer added (1.0 ml in total)
RNAlater®: if samples were stored in an RNAlater® solution or similar substance it is necessary to remove it before proceeding with RNA extraction. Centrifuge the sample at 10,000 rpm for 1 min and pipette the supernatant. Add 1 ml of PB pH 8.0, vortex the sample for 10 s to make sure the soil is washed by the buffer, and centrifuge it again at 10,000 rpm for 1 min Remove the supernatant completely and proceed with normal extraction.
Extra dry soil: for extra dry soil (< 5% WC) it is recommended to add some (up to 250 µl) PB pH 8.0 in addition to the usual amount of 375 µl during the first bead beating processing. The reason is that in dry soils some of the extraction buffer is inevitably absorbed in the soil and cannot be recovered afterwards. This can lead to low recovery volumes from the bead beating process which could make it hard to separate the aqueous phase from the phenolic phase and eventually result in reduced yields. After the first processing the soil is wet enough and no further additions are required.
pH of the extraction buffers: The phenol and the extraction buffers used in this protocol are all at pH 8.0. It has been shown that performing the extraction at pH 5.5 yields significantly fewer humic substances in the extractant and is recommended for soils rich in organic material. Using such low pH, however, will also significantly decrease the amount of DNA yield (while not affecting RNA yield). Low pH extraction might therefore not be suitable if DNA is also to be analysed. For more information see Mettel et al. 2010.
Number of bead beating processing: for most soil types (especially if they’re not too clayish), one time processing should be sufficient to obtain enough NA for further applications. The following two repetitions are meant not only to increase yields but also to reduce the bias associated with the susceptibility of different cell types to lysis. Feinstein et al. 2009 found that DNA extraction yields kept increasing even after the 6th bead beating repetition, and that community structure differed somewhat in each repetition.
Amount of crude extract to use for RNA purification: this depends mostly on the colour of the crude extract, and its quantity and purity as measured spectrophotometrically. In addition, the decision should take into account the final volume of the RNA .Between 20-100 µl may be used depending on colour, purity and amount of NA.
Quantification: it’s not possible to precisely quantify total NA using a spectrophotometer because DNA and RNA require different multiplication factors, but this should nevertheless give a sensible estimation. More precise quantifications of DNA and RNA can be obtained using Invitrogen’s PicoGreen™ and RiboGreen™, respectively. Impurities, especially humic substances, greatly interfere with spectrophotometeric quantification, though PicoGreen™ and RiboGreen™ tend to be less sensitive.
Three steps purification procedure: purification with phenol/chloroform and then chloroform should yield pure enough samples for most applications. Adding an additional purification step with only phenol prior to the phenol/chloroform purification (step 8) will ensure high TNA purity while only minimally compromising the yield.
For recipes of the solutions which need to be prepared see: Buffers and Solutions for TNA Extractions.pdf under ‘Figures’.
Before you begin
High yields of high quality DNA and RNA for downstream enzymatic reactions.
Solutions for TNA Extraction: Solutions for TNA Extraction
Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions. Roey Angel, Peter Claus, and Ralf Conrad. The ISME Journal 6 (4) 847 - 862 10/11/2011 doi:10.1038/ismej.2011.141
Roey Angel, Max Planck Institute for Terrestrial Microbiology
Correspondence to: Roey Angel ([email protected])
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.046. Originally published online 23 October 2012.