Improving Griffith's protocol for co-extraction of microbial DNA and RNA in adsorptive soils

Highlights

• We present an improved extraction protocol to increase nucleic acid yields from soil.

• Addition of an adsorption-site competitor released nucleic acids bound to soil.

• The protocol improved extraction efficiency from low biomass and high clay soils.

• This approach allowed quantitative transcriptional analysis in adsorptive soils.

• Microbial community diversity was analysed in low biomass/high clay soils.

Abstract

Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved extraction protocol that was optimized by: i) including an adsorption-site competitor prior to cell lysis to decrease adsorption of nucleic acids to soil particles, and ii) optimizing the PEG concentration used for nucleic acid precipitation. The extraction efficiency was determined using quantitative real-time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca²⁺ in combination with high pH. Modifications to the co-extraction protocol improved nucleic acid extraction efficiency from all adsorptive soils and were successfully validated by DGGE analysis of the indigenous community based on 16S rRNA gene and transcripts in soils representing low biomass and/or high clay content. This new approach reveals a robust co-extraction protocol for a range of molecular analysis of diverse soil environments.

Introduction

The current understanding of complex microbial communities and our ability to study them is greatly attributed to the continued development of reliable molecular tools for nucleic acid-based analysis. While the depth of resolution and sensitivity of these tools keep improving, they remain dependent on our ability to extract nucleic acids of sufficient quantity and quality for downstream analysis. Particularly, isolation of DNA and RNA from heterogeneous matrices like soil is challenging. Although a standardized method for DNA extraction from soil has recently been proposed (Petric et al., 2011), there is still no published method allowing high and uniform extraction efficiencies from all soil types. Extraction of RNA is even more challenging and particularly problematic for messenger RNA (mRNA) which constitutes only a small fraction of total soil RNA; hence, sufficient RNA extraction yields are crucial (Saleh-Lakha et al., 2011) to allow gene transcript analysis in soil systems.

Co-extraction of DNA and RNA allows for direct comparative studies when relating transcription rates to gene abundance (Bælum et al., 2008; Freitag and Prosser, 2009; Garcia et al., 2010; Nicolaisen et al., 2008; Paulin et al., 2010, 2011) and minimizes the bias created by using two different extraction procedures. Several protocols have been developed to extract either DNA or RNA from a variety of soils, whereas co-extraction protocols are rarely available (Costa et al., 2004; Griffiths et al., 2000; Hurt et al., 2001; McIlroy et al., 2009; Orsini and Romano-Spica, 2001; Töwe et al., 2010; Yu and Mohn, 1999). One of the most cited co-extraction protocols is the one originally developed by Griffiths et al. (2000) for analysis of 16S rRNA genes and transcripts; this protocol has since been optimized (Nicolaisen et al., 2008) and shown to provide a reliable assay for quantification of functional gene abundance and expression in agricultural soils (Bælum et al., 2008; Nicolaisen et al., 2008; Paulin et al., 2010, 2011), although extraction efficiencies vary greatly depending on soil type.

One major limitation preventing efficient extraction of nucleic acids from soil matrices is the ability of surface-reactive soil fractions such as clay and humic substances to bind nucleic acids (Pietramellara et al., 2009; Saeki et al., 2011), thereby causing a reduction in nucleic acid extraction yields (Frostegård et al., 1999; Volossiouk et al., 1995). Introducing certain molecules as adsorption site competitors prior to lysis of the soil microorganisms has been shown to largely increase extraction yields of DNA in certain soils (Hoshino and Matsumoto, 2005, 2007). However, it is unclear if this effect would be seen in other soil types having different physical and chemical properties, and in particular if it would also benefit extraction of RNA, as this has not previously been addressed. Adsorption of DNA to soil components has been studied previously with respect to protection from degradation (Lorenz and Wackernagel, 1987; Romanowski et al., 1991) and its influence on transformation rates of soil bacteria (Chamier et al., 1993). Yet, even though adsorption has an effect on nucleic acid extraction efficiency from some matrices (Barton et al., 2005; Yankson and Steck, 2009), there still remains a lack of information regarding the direct consequences of a soil's adsorption capacity on nucleic acid extraction recovery.

Another important consideration is co-extraction of inhibitory compounds, e.g. humic acids from soil. Downstream PCR-based methods are extremely sensitive to these inhibitors and even small amounts can negatively affect enzymatic processes leading to erroneous results (Jacobsen, 1995; Lloyd et al., 2010; Schneider et al., 2009; Tebbe and Vahjen, 1993). It has been reported that polyethylene glycol (PEG) precipitation is more effective for removal of humic acids than isopropanol and that lowering PEG concentrations down to 5–10% may reduce co-extraction of humic acids without significant loss of DNA yields (Arbeli and Fuentes, 2007; La Montagne et al., 2002). However, low concentrations of PEG reduce recovery of shorter nucleic acids (Lis, 1980) which could be problematic when extracting mRNA; hence, information on the influence of PEG concentration on the RNA yield should be investigated.

In the present study we first investigated DNA adsorption in nine different soils, representing a range of different chemical, physical and biological characteristics, and tested the effectiveness of adding salmon sperm DNA as an adsorption-site competitor for improved recovery of both RNA and DNA fractions of the indigenous microbial community. Based on these results, we developed an improved version of the Griffith DNA and RNA co-extraction protocol including the adsorption-site competitor and a column-based purification step. DNA and RNA yields were evaluated by quantitative real-time PCR (qPCR) by comparing the abundance of tfdA genes and transcripts in soils inoculated with the MCPA-degrading strain Cupriavidus necator AEO106 (pRO101). We also tested the effect of PEG concentration on detection of selected functional genes and transcripts. The improved protocol was finally verified by DGGE analysis of 16S rRNA genes and gene transcripts representing the indigenous microbial community in non-inoculated soils.