Zr soil fecal rna microprep kit

Manufactured by Zymo Research

Sourced in United States

The ZR Soil/Fecal RNA MicroPrep™ kit is a laboratory tool designed to efficiently extract and purify RNA from soil and fecal samples. It provides a straightforward method for isolating high-quality RNA from these complex sample types.

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ZR RNA MicroPrep kit (22 citations) RNA MicroPrep kit (17 citations) ZR RNA MicroPrep (9 citations) ZR Soil/Fecal RNA MicroPrep (6 citations) MicroPrep kits (4 citations) Zymo Research Soil/Fecal RNA kit (3 citations)

6 protocols using zr soil fecal rna microprep kit

Anaerobic Digester Total RNA Extraction

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Total RNA from samples of anaerobic digesters (A–J) was extracted using the ZR Soil/Fecal RNA MicroPrep™ kit (Zymo Research Europe GmbH, Freiburg, Germany), following the manufacturer's instructions. Remaining DNA within RNA extracts was removed using the Turbo DNA‐free™ kit (Ambion^® by Thermo Fisher Scientific GmbH, Dreieich, Germany). The success of the DNase treatment was confirmed by standard polymerase chain reaction (PCR) on 16S and 28S rRNA gene fragments, which were visualized by gel electrophoresis. RNA concentrations were measured spectrophotometrically at 260 nm (NanoDrop 2000 Spectrophotometer, Thermo Fisher Scientific GmbH). For cDNA synthesis from total RNA, a standard reverse transcription (RT)‐PCR (Green and Sambrook, 2012) was performed using SuperScript™ III Reverse Transcriptase and random hexamer primers (Thermo Fisher Scientific GmbH). Approximately 200 ng of total RNA isolated from anaerobic digesters were used as template for each RT‐PCR reaction.

Langer S.G., Gabris C., Einfalt D., Wemheuer B., Kazda M, & Bengelsdorf F.R. (2019). Different response of bacteria, archaea and fungi to process parameters in nine full‐scale anaerobic digesters. Microbial Biotechnology, 12(6), 1210-1225.

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Primer Extension Analysis of R. jostii GFP

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The R. jostii TMP1 bacteria harbouring the pART3-5′UTR-gfp were cultivated in minimal medium containing 0.05% of TTMP, until the OD₆₀₀ reached 0.5. In total, 1 mL of biomass was then collected by centrifugation (5 min, 16,100× g). The total RNA was isolated using ZR Soil/Fecal RNA MicroPrep kit (Zymo Research Corporation, Irvine, CA, USA). The 5′ end of the DNA primer (P_GFP_R), complementary to the EGFP gene sequence, was labelled with [γ-³²P]-ATP (Amersham Biosciences, Cleveland, OH, USA), using T4 polynucleotide kinase (Thermo Fisher Scientific). Then, the primers were separated from the labelled nucleotides by precipitation with ethanol in the presence of 2 M of ammonium acetate. The primer extension analysis (Sanger et al., 1977) was performed on the total RNA extracted from R. jostii harbouring the pART3-5‘UTR-gfp, under conditions of primer excess, using the avian myeloblastosis virus (AMV) reverse transcriptase (Thermo Fisher Scientific), as described by Truncaite et al. [45 (link)]. The reaction products were analysed on a 6% denaturing polyacrylamide gel (8 M urea, TBE) and visualized using a Fujifilm FLA-5100 phosphorimager.

Stanislauskienė R., Kutanovas S., Kalinienė L., Bratchikov M, & Meškys R. (2018). Tetramethylpyrazine-Inducible Promoter Region from Rhodococcus jostii TMP1. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(7), 1530.

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Nucleic Acid Extraction for Biogas Plant Samples

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DNA from biogas plants 1–7 was extracted using the ZR Fecal DNA MiniPrep™ Kit (Zymo Research; Irvine, USA). Cell lysis was performed with a high-speed cell disturber Precellys^® 24 Homogenisator (VWR, Germany) for 40 s at 5000 rpm. Additional cleanup of the isolated DNA was performed by Agencourt AMRure XP beads from Beckman Coulter (Brea, CA, USA). Isolated DNA was stored at − 20 °C until library preparation. RNA was isolated using the ZR Soil/Fecal RNA MicroPrep™ Kit (Zymo Research, Irvine, USA). Isolated RNA was stored at − 80 °C until further preparation steps. The qualities of nucleic acids were checked with the HS NGS and HS RNA Fragment Analysis Kit on a Fragment Analyzer (AATI, USA).
Nucleic acids from R1 and R2 samples were isolated according to a protocol published recently [7 (link)].

Grohmann A., Vainshtein Y., Euchner E., Grumaz C., Bryniok D., Rabus R, & Sohn K. (2018). Genetic repertoires of anaerobic microbiomes driving generation of biogas. Biotechnology for Biofuels, 11, 255.

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RNA Extraction from Soil Samples

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After testing multiple extraction kits and RNA preservation solutions, the ZR Soil/Fecal RNA MicroPrep kit (ZymoResearch Corporation) in combination with LifeGuard Soil Preservation solution was found to generate the highest RNA yields with the lowest inhibition to downstream reactions. All equipment was pre-treated with RNaseZap Wipes (Ambion, Grand Island, NY) and reagents and tubes were UV-sterilized for 30 min with the exception of S/F RNA Lysis Buffer (ZymoResearch Corporation). Soil samples were thawed on ice and centrifuged to remove the LifeGuard Soil Preservation solution (MO BIO Laboratories, Carlsbad, CA). The ZR manufacturer’s protocol was followed using 1.0 mL of S/F RNA Lysis solution and 5 min of bead beating. The full extraction volume was processed by sequential reloading of the Zymo-Spin IIIC column followed by RNA elution in 33 µL UV-sterilized Nuclease-free DEPC-treated H₂O (ISC BioExpress). Residual DNA was removed in a 35 min DNase reaction at 37°C as described previously (Neilson et al. 2010 (link)). RNA quality was confirmed by gel electrophoresis using the Alpha Imager system (Alpha Innotech, San Leandro, CA) and extracts were stored at −80°C.

Nelson K.N., Neilson J.W., Root R.A., Chorover J, & Maier R.M. (2015). Abundance and activity of 16S rRNA, amoA and nifH bacterial genes during assisted phytostabilization of mine tailings. International journal of phytoremediation, 17(5), 493-502.

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Extracting and Sequencing Fecal RNA

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A total of 250 mg of stools from the participants was used for total RNA extraction. For this, a ZR Soil/Fecal RNA MicroPrep™ kit (Zymo Research) was used following the manufacturer’s instructions, which not only allows one to obtain long RNAs but also makes it possible to obtain small RNAs. The RNA obtained was quantified with fluorometry using Quantus Fluorometer™ (Promega, Madison, WI, USA) and analyzed with capillary electrophoresis using Fragment Analyzer™ (Agilent Technologies, Santa Clara, CA, USA). For the qualification of samples, RNA had to have an RIN equal to or greater than 7.0. Subsequently, libraries were prepared using a TruSeq Small RNA Library Prep™ kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. The libraries were quantified with fluorometry using the Quantus Fluorometer™ (Promega, USA) and analyzed with capillary electrophoresis using Agilent 2100 Bioanalyzer™ (Agilent Technologies, USA). Finally, sequencing was performed with HiSeq 50SE (Illumina, USA) according to the manufacturer’s instructions.

Morales C., Arias-Carrasco R., Maracaja-Coutinho V., Seron P., Lanas F., Salazar L.A, & Saavedra N. (2023). Differences in Bacterial Small RNAs in Stool Samples from Hypercholesterolemic and Normocholesterolemic Subjects. International Journal of Molecular Sciences, 24(8), 7213.

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RNA Extraction and Diarrhea Detection

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The ZR Soil/Fecal RNA Microprep™ kit (Zymo Research, California, U.S.A) was used for RNA extraction according to the manufacturer’s instructions. Quality and quantity of the extracted RNA was measured at 260 and 280 nm using a NanoDrop™ 2000 Spectrophotometer, a concentration > 1.8 was considered good enough for subsequent steps. Reverse transcription was done using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer’s instructions. Synthesized cDNA was amplified using the Seeplex® Diarrhea-V ACE Detection kit (Seegene, Seoul, Republic of Korea) according to the manufacturer’s instructions. Amplicons were visualized using Ultraviolet irradiation after electrophoresis on a 3% agarose gel. Positive, negative and internal controls provided by the kit manufacturer were included in every run.

Philip C.O., Koech M., Kipkemoi N., Kirera R., Ndonye J., Ombogo A., Kirui M., Kipkirui E., Danboise B., Hulseberg C., Bateman S., Flynn A., Swierczewski B., Magiri E, & Odundo E. (2019). Evaluation of the performance of a multiplex reverse transcription polymerase chain reaction kit as a potential diagnostic and surveillance kit for rotavirus in Kenya. Tropical Diseases, Travel Medicine and Vaccines, 5, 12.

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