A sequential co-extraction method for DNA, RNA and protein recovery from soil for future system-based approaches

Abstract

A co-extraction protocol is presented that sequentially isolates core biopolymer fractions (DNA, RNA, protein) from edaphic microbial communities. In order to confirm compatibility with downstream analyses, bacterial T-RFLP profiles were generated from the DNA-and RNA-derived fractions of an arid-based soil, with metaproteomics undertaken on the corresponding protein fraction. were transferred to sterile 2 ml screw cap tubes (WhiteSci, Gauteng, SA). Ten grams was required for Soil type-A, while only 1 g of soil was required to recover sufficient DNA, RNA and protein from Soil type-B. Thereafter, 0.5 ml of a 10% (w/v) hexadecyltrimethylammonium 3 Sigma) was added to each 2 ml sample tube. Each tube was also supplemented with 0.25 g of zirconia beads (0.1 and 0.5 mm; BioSpec, Bartlesville, OK, USA). Cell lysis was achieved by bead beating, with the aqueous phase pooled and phenol removed by a chloroformisoamyl alcohol (24:1) phase separation (Fig. 1A) Total nucleic acids were precipitated by 30% polyethylene glycol 8000 (Sigma) -1.6M NaCl at 10°C for 2 h with 70% (v/v) ice-cold ethanol wash steps (twice). KeywordsThe organic phase containing the protein was also pooled and retained in a separate tube at 4°C. Residual liquid was removed from the original sample tubes containing the soil and 0.5 ml of fresh 1% sodium dodecyl sulphate (SDS; Merck) extraction buffer (10 mM Tris, 5 mM MgCl 2 ; pH 8), supplemented with protease inhibitor cocktail (10 μl/ml; Sigma) was added (Fig. 1A). A second bead-beating extraction step was applied with subsequent centrifugation and benzonase treatment (250U/μl; Sigma) undertaken on the pooled supernatants to remove any remaining nucleic acids. Thereafter, an additional phenol:chloroform:isoamyl alcohol (25:24:1; pH 8) step was applied to partition the organic phase, which was then added to the organic fraction from the first extraction step (Fig. 1A). Five volumes of 0.1M ammonium acetate in methanol was used to precipitate the protein (-20°C, overnight) with additional washing steps using ice-cold methanol (twice) and acetone (80% v/v; twice). The resulting pellets were resolubilised in 6M guanidine buffer supplemented with 10mM DTT 10mM Tris 5mM CaCL 2 (pH 8) with 1 min of sonication (sonicator bath VWR USC2600).Successful co-extraction of DNA and RNA was confirmed and quantified using a Nanodrop 1000 spectrophotometer (NanoDrop Products, Wilmington, DE, USA) and gel electrophoresis ( Step 1 Initial sample preparationStep 4 T-RFLP communityStep 5 Shotgun label-free metaproteomics 1 2 3 4 5 6 7 8 9 10Choroform:isoamyl alcohol (24:1) stepStep 2 Step 3 Isolation of protein fraction were identified, with 48 RNA-based OTUs recorded (Fig. 1C). These results were consistent with recent studies where commercial DNA extraction kits were employed (Makhalanyane et al. 2013; Stomeo et al., 2013), suggesting that representative phylogenetic information was recovered from oligotrophic soil using this approach. We noted that 21 OTUs were unique for the RNA-derived cDNA samples, confirmin...

Diel-scale temporal dynamics recorded for bacterial groups in Namib Desert soil

“…Total RNA was recovered from desert soil samples using an modified phenol-chloroform extraction technique84 with quantification using a Nanodrop 1000 spectrophotometer (NanoDrop Products, Wilmington, DE, USA). RNA preparations were treated with RQ1 RNase-Free (Promega) to remove contaminating DNA, with subsequent purification (RNeasy; Qiagen, GmbH, Hilden, Germany) and reverse transcription (Supersript ® III first strand synthesis; Invitrogen).…”

“…RNA preparations were treated with RQ1 RNase-Free (Promega) to remove contaminating DNA, with subsequent purification (RNeasy; Qiagen, GmbH, Hilden, Germany) and reverse transcription (Supersript ® III first strand synthesis; Invitrogen). Bacterial T-RFLP analysis of the cDNA products was done using 16S rRNA gene PCR primers 341 F (5′-FAM-CCTACGGGAGGCAGCAG-3′) and 908 R (5′-CCGTCAATTCCTTTRAGTTT-3′)84. Thermal cycling conditions were 94 °C for 5 min; 30 cycles of 94 °C for 30S, 55 °C for 20 s and 72 °C for 105 s; 72 °C for 10 min.…”

Namib Desert edaphic bacterial, fungal and archaeal communities assemble through deterministic processes but are influenced by different abiotic parameters

“…Soil metagenomic DNA (mDNA) was extracted using a modified phenol-chloroform extraction (Gunnigle et al 2014). In brief, 250 mg of 0.1 mm and 0.5 mm zirconia beads were added to sterile 2 ml screw cap tubes, followed by the addition of 1 g of soil.…”

A robust, cost‐effective method for DNA, RNA and protein co‐extraction from soil, other complex microbiomes and pure cultures

“…The protocol is based on the phenol-chloroform method of Griffiths, Whiteley, O'Donnell, and Bailey (2000), but is scaled up and modified to allow for the co-extraction of DNA, RNA and proteins from soil in sufficient yields to allow downstream analyses using meta-omics tools. Gunnigle, Ramond, Frossard, Seeley, and Cowan (2014) also reported on the co-extraction of DNA, RNA and proteins from soil samples, where they first recover nucleic acids prior to further bead beating to increase protein recovery yields. Feinstein, Sul, and Blackwood (2009), however, demonstrated that microbial DNA continues to be isolated from soil during consecutive bead beating regimes.…”