Abstract
A low-cost disruption of jute (Corchorus olitorius) rhizospheric microorganisms (Aspergillus niger and Arthrobacter sp.) with naturally occurring sand for extracting total RNA using modified QIAGEN RNeasy Mini Kit protocols is described. The method yielded distinct 18S and 28S, and 16S and 23S subunits of the fungal and bacterial ribosomal RNAs, respectively, on formaldehyde agarose gels. Total RNA extracted using this method was suitable for reverse transcription PCR to study the differential expression of mRNAs under the stress of herbicides. Primers OPM 1 and OPP 15 were effective in identifying upregulated transcripts in glyphosate and paraquat stressed A. niger. Similarly, primers OPP 13 and OPR 7 were effective in identifying upregulated transcripts in glyphosate-stressed Arthrobacter sp. This is the first report to show that natural sand can be combined with QIAGEN RNeasy Mini Kits for the extraction of total RNA from rhizospheric microorganisms. Novelty of the present method lies in the grinding of samples without the need of liquid nitrogen and then direct purification of RNA using modified QIAGEN RNeasy Mini Kit protocols, without chloroform extraction.
References
Atkinson NJ, Lilley CJ, Urwin PE (2013) Identification of genes involved in the response of Arabidopsis to simultaneous biotic and abiotic stresses. Plant Physiol 162:2028–2041. doi:10.1104/pp. 113.222372
Burden D (2012) Guide to the disruption of biological samples—2012. Random Prim 25:1–25
Chan C, Teo S, Ho C (2004) Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta). J Appl Phycol 16:297–301
De S, Kaur G, Roy A, Dogra G, Kaushik R, Yadav P, Singh R, Datta TK, Goswami SL (2010) A simple method for the efficient isolation of genomic DNA from lactobacilli isolated from traditional Indian fermented milk (dahi). Indian J Microbiol 50:412–418. doi:10.1007/s12088-011-0079-4
Francesconi A, Kasai M, Harrington SM, Beveridge MG, Petraitiene R, Petraitis V, Schaufele RL, Walsh TJ (2008) Automated and manual methods of DNA extraction for Aspergillus fumigatus and Rhizopus oryzae analyzed by quantitative real-time PCR. J Clin Microbiol 46:1978–1984. doi:10.1128/JCM. 02246-07
García-Nogales P, Serrano A, Secchi S, Gutiérrez S, Arís A (2010) Comparison of commercially-available RNA extraction methods for effective bacterial RNA isolation from milk spiked samples. Electron J Biotechnol. doi:10.2225/vol13-issue5-fulltext-10
Gelsthorpe AR, Sokol RJ (1997) Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues. Biotechniques 22:1082–1086
Hurt RA, Qiu X, Wu L, Roh Y, Palumbo AV, Tiedje JM, Zhou J (2001) Simultaneous recovery of RNA and DNA from soils and sediments simultaneous recovery of RNA and DNA from soils and sediments. Appl Environ Microbiol 67:4495–4503. doi:10.1128/AEM.67.10.4495
Kharbikar LL, Majumdar B, Ghorai AK, Chakraborty L, Sarkar D, Mahapatra BS (2009) Herbicides resistant soil microorganisms isolated from the rhizosphere of jute (Corchorus spp.). In: Anonymous (ed) Proc. Int. Conf. Emerg. Trends Prod. Process. Util. Nat. Fibres, Souvenir. Indian Society for Cotton Improvement and Indian Fibre Society, pp 140–147
Leite GM, Magan N, Medina Á (2012) Comparison of different bead-beating RNA extraction strategies: an optimized method for filamentous fungi. J Microbiol Methods 88:413–418. doi:10.1016/j.mimet.2012.01.011
Lim CSY, Tung CH, Rosli R, Chong PP (2008) An alternative Candida spp. cell wall disruption method using a basic sorbitol lysis buffer and glass beads. J Microbiol Methods 75:576–578. doi:10.1016/j.mimet.2008.07.026
Mangan JA, Sole KM, Mitchison DA, Butcher PD (1997) An effective method of RNA extraction from bacteria refractory to disruption, including mycobacteria. Nucleic Acids Res 25:675–676
Oh ET, So J-S (2003) A rapid method for RNA preparation from Gram-positive bacteria. J Microbiol Methods 52:395–398
Rittmann S, Holubar P (2014) Rapid extraction of total RNA from an anaerobic sludge biocoenosis. Folia Microbiol 59(2):127–132
Rosok O, Odeberg J, Rode M (1996) Solid-phase method for differential display of genes expressed in hematopoietic stem cells. Biotechniques 21:114–121
Sallau AB, Henriquez F, Nok AJ, Ibrahim S, Sommerville C, Roberts C (2013) Aspergillus niger -specific ribonucleic acid extraction method. J Yeast Fungal Res 4:58–62. doi:10.5897/JYFR2013.0107
Shu C, Sun S, Chen JJ, Zhou E (2014) Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani. Electron J Biotechnol 17:50–54. doi:10.1016/j.ejbt.2013.12.009
Singh BK, Walker A (2006) Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev 30:428–471. doi:10.1111/j.1574-6976.2006.00018.x
Sturtevant J (2000) Applications of differential-display reverse transcription-PCR to molecular pathogenesis and medical mycology. Clin Microbiol Rev 13:408–427
Vollmer W, Blanot D, de Pedro MA (2008) Peptidoglycan structure and architecture. FEMS Microbiol Rev 32:149–167. doi:10.1111/j.1574-6976.2007.00094.x
Zhang T, Breitbart M, Lee WH, Run J-Q, Wei CL, Soh SWL, Hibberd ML, Liu ET, Rohwer F, Ruan Y (2006) RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biol 4:e3. doi:10.1371/journal.pbio.0040003
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kharbikar, L.L., Majumdar, B. A low-cost disruption of rhizospheric microorganisms for the extraction of total RNA using modified RNeasy Mini Kit protocols. Ann Microbiol 65, 1797–1801 (2015). https://doi.org/10.1007/s13213-015-1052-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13213-015-1052-y