Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics


Metatranscriptomics—gene express profiling via DNA sequencing—is a powerful tool to identify genes that are actively expressed and might contribute to the phenotype of individual organisms or the phenome (the sum of several phenotypes) of a microbial community. Furthermore, metatranscriptome studies can result in extensive catalogues of genes that encode for enzymes of industrial relevance. In both cases, a major challenge for generating a high quality metatranscriptome is the extreme lability of RNA and its susceptibility to ubiquitous RNAses. The microbial community (the microbiome) of the cow rumen efficiently degrades lignocelullosic biomass, generates significant amounts of methane, a greenhouse gas twenty times more potent than carbon dioxide, and is of general importance for the physiological wellbeing of the host animal. Metatranscriptomes of the rumen microbiome from animals kept under different conditions and from various types of rumen-incubated biomass can be expected to provide new insights into these highly interesting phenotypes and subsequently provide the framework for an enhanced understanding of this socioeconomically important ecosystem. The ability to isolate large amounts of intact RNA will significantly facilitate accurate transcript annotation and expression profiling. Here we report a method that combines mechanical disruption with chemical homogenization of the sample material and consistently yields 1 mg of intact RNA from 1 g of rumen-incubated biofuel feedstock. The yield of total RNA obtained with our method exceeds the RNA yield achieved with previously reported isolation techniques, which renders RNA isolated with the method presented here as an ideal starting material for metatranscriptomic analyses and other molecular biology applications that require significant amounts of starting material.

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H. Piao, L. Markillie, D. Culley, R. Mackie and M. Hess, "Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics," Advances in Microbiology, Vol. 3 No. 1, 2013, pp. 101-107. doi: 10.4236/aim.2013.31016.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] P. N. Hobson and C. S. Stewart, “The Rumen Microbial Ecosystem,” 2nd Edition, Blackie Academic & Professional, New York, 1997. doi:10.1007/978-94-009-1453-7
[2] A. N. Hristov, T. R. Callaway, C. Lee and S. E. Dowd, “Rumen Bacterial, Archaeal, and Fungal Diversity of Dairy Cows in Response to Ingestion of Lauric or Myristic Acid,” Journal of Animal Science, Vol. 90, No. 12, 2012, pp. 4449-4457. doi:10.2527/jas.2011-4624
[3] T. A. McAllister, H. D. Bae, G. A. Jones and K. J. Cheng, “Microbial Attachment and Feed Digestion in the Rumen,” Journal of Animal Science, Vol. 72, No. 11, 1994, pp. 3004-3018.
[4] E. M. Rubin, “Genomics of Cellulosic Biofuels,” Nature, Vol. 454, No. 7026, 2008, pp. 841-845.
[5] D. B. Wilson, “Microbial Diversity of Cellulose Hydrolysis,” Current Opinion in Microbiology, Vol. 14, No. 3, 2011, pp. 259-263. doi:10.1016/j.mib.2011.04.004
[6] R. Mackelprang, M. P. Waldrop, K. M. DeAngelis, M. M. David, K. L. Chavarria, S. J. Blazewicz, E. M. Rubin and J. K. Jansson, “Metagenomic Analysis of a Permafrost Microbial Community Reveals a Rapid Response to Thaw,” Nature, Vol. 480, No. 7377, 2011, pp. 368-371. doi:10.1038/nature10576
[7] M. Allgaier, A. Reddy, J. I. Park, N. Ivanova, P. D’Haeseleer, S. Lowry, R. Sapra, T. C. Hazen, B. A. Simmons, J. S. VanderGheynst and P. Hugenholtz, “Targeted Discovery of Glycoside Hydrolases from a Switchgrass—Adapted Compost Community,” PloS One, Vol. 5, No. 1, 2010, pp. 1-9.
[8] M. Hess, A. Sczyrba, R. Egan, T. W. Kim, H. Chokhawala, G. Schroth, S. Luo, D. S. Clark, F. Chen, T. Zhang, R. I. Mackie, L. A. Pennacchio, S. G. Tringe, A. Visel, T. Woyke, Z. Wang and E. M. Rubin, “Metagenomic Discovery of Biomass-Degrading Genes and Genomes from Cow Rumen,” Science, Vol. 331, No. 6010, 2011, pp. 463-467.
[9] P. B. Pope, S. E. Denman, M. Jones, S. G. Tringe, K. Barry, S. A. Malfatti, A. C. McHardy, J. F. Cheng, P. Hugenholtz, C. S. McSweeney and M. Morrison, “Adaptation to Herbivory by the Tammar Wallaby Includes Bacterial and Glycoside Hydrolase Profiles Different from Other Herbivores,” Proceedings of the National Academy of Sciences of the USA, Vol. 107, No. 33, 2010, pp. 14793-14798. doi:10.1073/pnas.1005297107
[10] G. Suen, J. J. Scott, F. O. Aylward, S. M. Adams, S. G. Tringe, A. A. Pinto-Tomas, C. E. Foster, M. Pauly, P. J. Weimer, K. W. Barry, L. A. Goodwin, P. Bouffard, L. Li, J. Osterberger, T. T. Harkins, S. C. Slater, T. J. Donohue and C. R. Currie, “An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity,” PLoS Genetics, Vol. 6, No. 9, 2010, Article ID: e1001129.
[11] P. Narasingarao, S. Podell, J. A. Ugalde, C. BrochierArmanet, J. B. Emerson, J. J. Brocks, K. B. Heidelberg, J. F. Banfield and E. E. Allen, “De Novo Metagenomic Assembly Reveals Abundant Novel Major Lineage of Archaea in Hypersaline Microbial Communities,” The ISME Journal, Vol. 6, No. 1, 2012, pp. 81-93. doi:10.1038/ismej.2011.78
[12] F. Warnecke and M. Hess, “A Perspective: Metatranscriptomics as a Tool for the Discovery of Novel Biocatalysts,” Journal of Biotechnology, Vol. 142, No. 1, 2009, pp. 91-95. doi:10.1016/j.jbiotec.2009.03.022
[13] M. Qi, P. Wang, N. O’Toole, P. S. Barboza, E. Ungerfeld, M. B. Leigh, L. B. Selinger, G. Butler, A. Tsang, T. A. McAllister and R. J. Forster, “Snapshot of the Eukaryotic gene Expression in Muskoxen Rumen—A Metatranscriptomic Approach,” PLoS One, Vol. 6, No. 5, 2011, p. e20521.
[14] K. C. McGrath, S. R. Thomas-Hall, C. T. Cheng, L. Leo, A. Alexa, S. Schmidt and P. M. Schenk, “Isolation and Analysis of mRNA from Environmental Microbial Communities,” Journal of Microbiological Methods, Vol. 75, No. 2, 2008, pp. 172-176.
[15] J. A. Gilbert, D. Field, Y. Huang, R. Edwards, W. Li, P. Gilna and I. Joint, “Detection of Large Numbers of Novel Sequences in the Metatranscriptomes of Complex Marine Microbial Communities,” PLoS One, Vol. 3, No. 8, 2008, p. e3042. doi:10.1371/journal.pone.0003042
[16] J. Frias-Lopez, Y. Shi, G. W. Tyson, M. L. Coleman, S. C. Schuster, S. W. Chisholm and E. F. Delong, “Microbial Community Gene Expression in Ocean Surface Waters,” Proceedings of the National Academy of Sciences of the USA, Vol. 105, No. 10, 2008, pp. 3805-3810. doi:10.1073/pnas.0708897105
[17] R. S. Poretsky, I. Hewson, S. Sun, A. E. Allen, J. P. Zehr and M. A. Moran, “Comparative Day/Night Metatranscriptomic Analysis of Microbial Communities in the North Pacific Subtropical Gyre,” Environmental Microbiology, Vol. 11, No. 6, 2009, pp. 1358-1375. doi:10.1111/j.1462-2920.2008.01863.x
[18] R. S. Poretsky, S. Gifford, J. Rinta-Kanto, M. Vila-Costa and M. A. Moran, “Analyzing Gene Expression from Marine Microbial Communities Using Environmental Transcriptomics,” Journal of Experimental Medicine, Vol. 18, No. 24, 2009, p. 1086.
[19] C. Bera-Maillet, P. Mosoni, A. Kwasiborski, F. Suau, Y. Ribot and E. Forano, “Development of a RT-qPCR Method for the Quantification of Fibrobacter Succinogenes S85 Glycoside Hydrolase Transcripts in the Rumen Content of Gnotobiotic and Conventional Sheep,” Journal of Microbiological Methods, Vol. 77, No. 1, 2009, pp. 8-16. doi:10.1016/j.mimet.2008.11.009
[20] S. Kang, S. E. Denman, M. Morrison, Z. Yu and C. S. McSweeney, “An Efficient RNA Extraction Method for Estimating Gut Microbial Diversity by Polymerase Chain Reaction,” Current Microbiology, Vol. 58, No. 5, 2009, pp. 464-471. doi:10.1007/s00284-008-9345-z
[21] P. Wang, M. Qi, P. Barboza, M. B. Leigh, E. Ungerfeld, L. B. Selinger, T. A. McAllister and R. J. Forster, “Isolation of High-Quality Total RNA from Rumen Anaerobic Bacteria and Fungi, and Subsequent Detection of Glycoside Hydrolases,” Analytical Biochemistry, Vol. 57, No. 7, 2011, pp. 590-598. doi:10.1139/w11-048
[22] P. Chomczynski and N. Sacchi, “Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction,” Analytical Biochemistry, Vol. 162, No. 1, 1987, pp. 156-159.
[23] P. Chomczynski and N. Sacchi, “The Single-Step Method of RNA Isolation by Acid Guanidinium ThiocyanatePhenol-Chloroform Extraction: Twenty-Something Years On,” Nature Protocols, Vol. 1, No. 2, 2006, pp. 581-585. doi:10.1038/nprot.2006.83
[24] S. Ramos, M. L. Tejido, M. J. Ranilla, M. E. Martinez, C. Saro and M. D. Carro, “Influence of Detachment Procedure and Diet on Recovery of Solid-Associated Bacteria from Sheep Ruminal Digesta and Representativeness of Bacterial Isolates as Assessed by Automated Ribosomal Intergenic Spacer Analysis-Polymerase Chain Reaction,” Journal of Dairy Science, Vol. 92, No. 11, 2009, pp. 5659-5668. doi:10.3168/jds.2009-2273
[25] N. Xu, Y. Li, Y. T. Zhao, L. Guo, Y. Y. Fang, J. H. Zhao, X. J. Wang, L. Huang and H. S. Guo, “Identification and Characterization of Small RNAs in the Hyperthermophilic Archaeon Sulfolobus Solfataricus,” PLoS One, Vol. 7, No. 4, 2012, p. e35306. doi:10.1371/journal.pone.0035306
[26] S. C. Li, W. C. Chan, M. R. Ho, K. W. Tsai, L. Y. Hu, C. H. Lai, C. N. Hsu, P. P. Hwang and W. C. Lin, “Discovery and Characterization of Medaka miRNA Genes by Next Generation Sequencing Platform,” BMC Genomics, Vol. 11, No. S4, 2010, p. S8. doi:10.1186/1471-2164-11-S4-S8
[27] M. A. Hernandez-Prieto, V. Schon, J. Georg, L. Barreira, J. Varela, W. R. Hess and M. E. Futschik, “Iron Deprivation in Synechocystis: Inference of Pathways, Non-Coding RNAs, and Regulatory Elements from Comprehensive Expression Profiling,” G3 (Bethesda), Vol. 2, No. 12, 2012, pp. 1475-1495.
[28] Q. Zhou, Z. Wang, J. Zhang, H. Meng and B. Huang, “Genome—Wide Identification and Profiling of MicroRNALike RNAs from Metarhizium Anisopliae during Development,” Fungal Biology, Vol. 116, No. 11, 2012, pp. 1156-1162. doi:10.1016/j.funbio.2012.09.001
[29] A. Schroeder, O. Mueller, S. Stocker, R. Salowsky, M. Leiber, M. Gassmann, S. Lightfoot, W. Menzel, M. Granzow and T. Ragg, “The RIN: An RNA Integrity Number for Assigning Integrity Values to RNA Measurements,” BMC Molecular Biology, Vol. 7, No. 3, 2006. doi:10.1186/1471-2199-7-3

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