Extraction of mRNA from Soil

Abstract

Here, we report an efficient method for extracting high-quality mRNA from soil. Key steps in the isolation of total RNA were low-pH extraction (pH 5.0) and Q-Sepharose chromatography. The removal efficiency of humic acids was 94 to 98% for all soils tested. To enrich mRNA, subtractive hybridization of rRNA was most efficient. Subtractive hybridization may be followed by exonuclease treatment if the focus is on the analysis of unprocessed mRNA. The total extraction method can be completed within 8 h, resulting in enriched mRNA ranging from 200 bp to 4 kb in size.Over the last decade, several methods have been reported for the extraction of environmental RNA (e.g., 11, 13, 32). These methods have been widely used to study the compositions and dynamics of microbial communities at the rRNA level (e.g., 19, 33). However, rRNA surveys provide no or only indirect information on the functional status of a microbial community. Therefore, monitoring the environmental expression of key genes of particular metabolic pathways, such as the genes for nitrogen fixation (nifH), nitrite reduction (nirS, nirK), ammonia oxidation (amoA), or methane oxidation (pmoA), received increasing attention (3,5,8,16,17,25). Most recently, new sequencing platforms, such as 454 pyrosequencing, allowed the metatranscriptome analysis of complex microbial communities (9, 10, 23, 31). However, it remains challenging to extract mRNA of high quality from soil for use in metatranscriptomics, due to the coextraction of humic acids and other organic compounds. These contaminants inhibit downstream analyses such as RNA amplification and reverse transcription-PCR, thus calling for the need of their quantitative removal. The low content of mRNA in total RNA extracts (1 to 5%) and their greater susceptibility to degradation by RNases than rRNA also hamper the efficient extraction of intact mRNA from soil (1, 2).Here, we report an efficient method for the extraction of total RNA and enrichment of mRNA from soils differing in their amounts and compositions of humic acids, including (i) rice paddy, (ii) grassland, (iii) agricultural, and (iv) forest soils. Assessments were made with regard to the (i) quantitative removal of humic acids, (ii) yield and integrity of total RNA, and (iii) size distribution of enriched mRNA. The optimized method allows the extraction of total RNA and enrichment of mRNA from multiple samples within 8 h. The procedural steps are described in their order of application (Fig. 1). Additional information on the procedural steps is given in the supplemental material.Extraction of total RNA. Samples from all four different soil types were processed in the same way. Fresh soil (0.5 g, wet weight) was suspended in 500 l of RNAlater (Ambion, Germany) and stored at 4°C overnight. Soil samples were pelleted at 20,000 ϫ g for 1 min, and the supernatants were discarded. No rRNA or other nucleic acids could be detected in the supernatants, indicating that the RNAlater treatment and subsequent centrifugation do not lead to the loss of RNA.The pelle...

Elucidating the impact of microbial community biodiversity on pharmaceutical biotransformation during wastewater treatment

“…DNA samples were prepared for shotgun metagenomic sequencing at the University of Michigan DNA Sequencing Core (details provided in Appendix S1) and sequenced on a 100‐cycle paired end run on a HiSeq 2500 (Illumina). RNA samples were prepared for shotgun metatranscriptomic sequencing by first enriching the mRNA from the total RNA extracts using the MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen, Carlsbad, CA) based on previous studies (He et al ., 2010; Mettel et al ., 2010). Individual libraries were prepared for each sample as for the DNA samples, and the samples were multiplexed using sample‐specific adaptors on a single lane of a HiSeq Flow Cell (Illumina).…”

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA

“…77, 2011 TARGETING hsp70 mRNA TO DETECT VIABLE C. PARVUM 6483 on May 9, 2018 by guest http://aem.asm.org/ made to our methods to detect lower numbers of oocysts in soil samples by reducing the influence of humic substance. For instance, mRNA extraction under low pH (pH 5.0) has been suggested to be essential to minimize the coextraction of humic acids from soil samples and to increase the stability of mRNA (47). The pH of the lysis buffer and phenol-chloroform mixture could be adjusted to 5.0 for soil samples with high humic acid content.…”

“…Although many soil RNA extraction procedures have been described (4,18,19,23,36,44,47,52), in practice, however, the efficient recovery of mRNA from soil remains a challenge. Three interfering factors may include the following: (i) mRNA is prone to degradation by RNase, which is ubiquitous in soil environments; (ii) soils are rich in humic substances, which could be coextracted with mRNA and inhibit the subsequent RT-PCR even at minute concentrations; and (iii) mRNA binds to naturally present soil components such as clay.…”

Metatrascriptomic Study of Microbes in Environment: A Community Function Based Approach

“…He et al [26] compared two methods of removing rRNA, i) rRNA-specific exonuclease treatment and ii) rRNA hybridization capturebased protocols for removal of rRNA as an approach of enriching mRNA prior to cDNA prperation. Comparison of two commercial kits, based on i) subtractive hybridization of rRNA (Ambion MICROB Express bacterial mRNA enrichment kit) and ii) rRNA specific exonuclease (Epicenter mRNA-ONLY prokaryotic mRNA isolation kit) were also compared [22]. Both of them observed and concluded that rRNA removal by subtractive hybridization yielded a better result with respect to degree of rRNA removal and quantitative mRNA yield.…”

“…However, due to extensive research on meta transcripts and technological advancements, it has now been possible to successfully isolate RNA from ecological samples [21,22]. Several commercial kits have been made available for extracting RNA from diverse ecological samples as summarized by Wang et al [15].…”