Microbenrich kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MICROBEnrich kit is a laboratory tool designed for the enrichment and purification of microbial RNA from complex samples. The kit utilizes selective hybridization and magnetic bead-based separation to efficiently capture and isolate microbial RNA, enabling researchers to focus their analysis on the microbial component of their samples.

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42 protocols using microbenrich kit

Bacterial RNA Enrichment from Mouse Spleens

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Once the optimal infection conditions were determined, a different set of BALB/c mice was infected with 0.1× LD₅₀ bacteria grown in SEM, and on day 5 postinfection, mouse spleens were harvested for total RNA extraction. Spleens were removed aseptically and stored in RNAlater (Ambion, Austin, TX) at −80°C until RNA extraction was conducted. Total RNA was extracted from 50 mg of each spleen using TRIzol (Invitrogen, Carlsbad, CA), subjected to RNase-free DNase set (Qiagen, Hilden, Germany) treatment according to the manufacturer’s instructions and purified using the RNeasy Minikit (Qiagen) as directed by the manufacturer.
Bacterial RNA was enriched from the total RNA extracted from mouse spleens using a method adapted from Mavromatis et al. (80 (link)). To eliminate host total RNA as well as bacterial and host rRNAs, 25 g of the extracted mouse spleen total RNA was processed with a MICROBEnrich kit (Ambion) according to the manufacturer’s instructions. The total RNA was then treated with the Ribo-Zero Gold rRNA Removal kit (Epidemiology) (Illumina, San Diego, CA) as directed by the manufacturer, to remove leftover host or bacterial rRNA. The quality and quantity of RNA obtained following bacterial RNA enrichment and rRNA depletion were determined with an Agilent 2100 Bioanalyzer and a LabChip 6000 RNA kit (Agilent Technologies, Santa Clara, CA).

Ghazali A.K., Firdaus-Raih M., Uthaya Kumar A., Lee W.K., Hoh C.C, & Nathan S. (2023). Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches. Microbiology Spectrum, 11(2), e03835-22.

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Isolation of Algal and Bacterial mRNA

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Two different approaches have been tested. The data presented in this manuscript has been generated from samples using the following strategy: The Oligotex mRNA kit (Qiagen, Hilden, Germany) was used to isolate the algal mRNA. For isolation of D. shibae mRNA, the MICROBEnrich kit (Ambion, Life Technologies, Darmstadt, Germany) was used to remove the eukaryotic rRNA, and the MICROBExpress kit (Ambion, Life Technologies, Darmstadt, Germany) was used to remove the prokaryotic rRNA, according to the manufacturer's instructions. A second approach resulted in an mRNA proportion of about 0.01%. These samples have therefore not been analyzed further: PolyATract System IV (Promega, Madsion, WI, USA) was used for isolation of algal mRNA according to the manufacturer's instructions. For isolation of the bacterial mRNA Ribo-Zero Magnetic Kit for both plant leaf and gram-negative bacteria (Epicenter, Madsion, WI, USA) were used according to the manufacturer's instructions. The final purification of the eluted mRNA was performed using ethanol precipitation.

Wang H., Tomasch J., Jarek M, & Wagner-Döbler I. (2014). A dual-species co-cultivation system to study the interactions between Roseobacters and dinoflagellates. Frontiers in Microbiology, 5, 311.

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Profiling Nitrogen Fixation Transcripts

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Strain A1501 was cultured for 5 h under nitrogen fixation and ammonium shock conditions as described previously (24 (link), 25 ). RNA was extracted using TRIzol LS reagent (Invitrogen, USA) following the manufacturer’s instructions. Host-cell RNA was depleted using a MICROBEnrich kit (Ambion, USA), and bacterial 23S and 16S rRNAs were subsequently depleted with a MICROBExpress bacterial mRNA enrichment kit (Ambion, USA). Total RNA-seq libraries were then constructed and sequenced using an Illumina HiSeq 2500 instrument with the paired-end method, and ncRNAs were predicted based on the mapping of read pairs by Tianjin Biochip Corporation (Tianjin, China).

Zhan Y., Deng Z., Yan Y., Zhang H., Lu C., Yang Z., Shang L., Huang Y., Lv F., Liu Y., Liu Y., Wang S., Chen S., Zhang X.X., Cheng Q, & Lin M. (2019). NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501. Applied and Environmental Microbiology, 85(14), e00762-19.

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RNA-seq of Anaplasma marginale in Cell Culture and Bovine Blood

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RNA was prepared from two biological replicates of A. marginale St. Maries strain maintained in ISE6 cell culture as previously described [13 (link),14 (link),24 (link)] and two biological replicates from infected bovine blood [21 (link)]. Samples were collected and processed using TRIzol (Invitrogen, Carlsbad, CA, USA) for RNA extraction, the MICROBEnrich kit (Ambion, Austin, TX, USA) to negatively select eukaryotic RNA and were normalized using Duplex-Specific thermostable Nuclease (DSN) (Illumina, San Diego, CA, USA) [21 (link),25 (link)]. Samples were sequenced with Illumina technology with 100 base pair reads [25 (link)]. The accession numbers for this RNA-seq study in the GenBank Sequence Read Archive (SRA) are SRP014580 and SRP014580.

Hammac G.K., Pierlé S.A., Cheng X., Scoles G.A, & Brayton K.A. (2014). Global transcriptional analysis reveals surface remodeling of Anaplasma marginale in the tick vector. Parasites & Vectors, 7, 193.

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Bacterial RNA Isolation and Sequencing

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RNA isolation protocol was previously described (Subashchandrabose et al., 2014 (link)). Briefly, samples were treated with proteinase K and total RNA was isolated using Qiagen RNAeasy minikit. Turbo DNase kit (Ambion) was used to remove contaminating DNA. Bacterial content of patient samples was enriched using MICROBEnrich kit (Ambion), which depletes RNA of eukaryotic mRNA and rRNA. Library preparation and sequencing was performed by University of Michigan sequencing core. ScriptSeq Complete Kit (Bacteria) library kit was used to both deplete samples of bacterial rRNA and to construct stranded cDNA libraries from the rRNA-depleted RNA (Table 3, Table 4). While the original in vitro samples submitted for sequencing were not treated with MICROBEnrich kit, we have since performed extensive testing with two different clinical UTI strains (HM86 and HM56) to show that treatment with the kit does not affect the measured gene expression (Figure 1—figure supplement 5, Supplementary file 1). All samples were sequenced using Illumina HiSeq2500 (single end, 50 bp read length).

Sintsova A., Frick-Cheng A.E., Smith S., Pirani A., Subashchandrabose S., Snitkin E.S, & Mobley H. (2019). Genetically diverse uropathogenic Escherichia coli adopt a common transcriptional program in patients with UTIs. eLife, 8, e49748.

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Bacterial RNA Enrichment from Host

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Bacterial RNA was enriched from the total RNA by using MICROBEnrich kit (Ambion Inc., USA; AM1901) as per the manufacturer’s protocol. Here, hybridization capture technology was used to remove human, mouse, and rat RNA (both mRNA and rRNA) from complex host-bacterial RNA populations, leaving behind enriched microbial total RNA. In the first step of the procedure, host-bacterial total RNA is incubated with a mixture of capture oligonucleotides that bind the mammalian 18S and 28S rRNAs and polyadenylated RNAs. Next, the rRNA/oligo nucleotide hybrids and all polyadenylated mRNAs are removed from the mixture with oligonucleotide-derivatized magnetic beads. To ensure complete removal of eukaryotic mRNAs, complementary DNA was constructed with oligo-d(T) primers and polymerase chain reaction for the mouse GAPDH gene was executed and checked.

Velmurugan G., Ramprasath T., Swaminathan K., Mithieux G., Rajendhran J., Dhivakar M., Parthasarathy A., Babu D.D., Thumburaj L.J., Freddy A.J., Dinakaran V., Puhari S.S., Rekha B., Christy Y.J., Anusha S., Divya G., Suganya K., Meganathan B., Kalyanaraman N., Vasudevan V., Kamaraj R., Karthik M., Jeyakumar B., Abhishek A., Paul E., Pushpanathan M., Rajmohan R.K., Velayutham K., Lyon A.R, & Ramasamy S. (2017). Gut microbial degradation of organophosphate insecticides-induces glucose intolerance via gluconeogenesis. Genome Biology, 18, 8.

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Isolation and RNA-Seq Analysis of B. bacilliformis

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Upon shifting B. bacilliformis for the designated time periods, cells were either harvested directly into one volume of RNAlater solution (Thermo Fisher) or centrifuged at 10,000 x g at room temperature for 2 min, after which the pellet was resuspended in a volume of RNAlater. The cells were incubated at room temperature for 1 h then frozen at -80°C for ≥ 2 h. The cells were thawed, centrifuged at 10,000 x g at 4°C for 10 min, and resuspended in 1 ml of TRI Reagent (Sigma-Aldrich; St. Louis, MO). The cells were incubated at room temperature for 1 h then frozen at -80°C for ≥ 2 h. Finally, cells were thawed and total RNA and genomic DNA isolation were done as previously described [36 (link)]. Total RNA pools from human blood infections were globin-depleted using a GLOBINclear kit (Ambion) according to manufacturer’s specifications. HUVE, HB37, and HBBG samples were enriched for bacterial RNA using a MICROBEnrich kit (Ambion). RNA (1 μg) from three independent biological replicates of each condition was sent to the Yale Center for Genomic Analysis (Pl25, Pl30, Pl37, pH06, pH07, and pH08 samples) or GENEWIZ (PlBG, HUVE, HB37, and HBBG samples) for bacterial rRNA depletion, stranded-library preparation, and HiSeq2500 (Illumina; San Diego, CA) 2x150 bp sequencing.

Wachter S., Hicks L.D., Raghavan R, & Minnick M.F. (2020). Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host. PLoS Neglected Tropical Diseases, 14(11), e0008671.

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Host-Microbiome RNA Extraction Protocol

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Approximately 200 mg of tissue sample was placed in 1.5 ml RNAlater buffer (Ambion) and snap-frozen in liquid nitrogen. RNA extraction was performed as described previously (Giannoukos et al., 2012 (link)). In brief, at time of extraction, samples were centrifuged for 10 min at 16,000 g at room temperature. Pellets were resuspended in 150 µl lysis buffer (Tris/HCl, pH 8, 1 mM EDTA, 15 mg/ml Lysozyme [Sigma-Aldrich], and 15 µl of 20 mg/ml proteinase K) and incubated at room temperature for 10 min with brief mixing every 2 min. After addition of 1.2 ml QIAGEN RLT buffer containing 1% vol/vol β-mercaptoethanol, 1 ml of 0.1-mm glass beads (BioSpec) was added, and samples were homogenized in a FastPrep at setting 5 (four pulses of 20 s). Samples were kept on ice for 1 min between pulses. Lysates were then homogenized with a QIAshredder spin column, and RNA was isolated using the AllPrep mini kit (QIAGEN), according to the manufacturer’s protocols, which included an on-column digestion with DNase I. 25 µg RNA from each tissue sample was processed with the MICROBEnrich kit (Ambion), and 5 µg of processed RNA was further depleted of rRNAs using the Meta-Bacteria RiboZero rRNA removal kit (Epicentre). The final samples consisted of a mix of host and microbial mRNAs in a 2:1 ratio.

Bongers G., Pacer M.E., Geraldino T.H., Chen L., He Z., Hashimoto D., Furtado G.C., Ochando J., Kelley K.A., Clemente J.C., Merad M., van Bakel H, & Lira S.A. (2014). Interplay of host microbiota, genetic perturbations, and inflammation promotes local development of intestinal neoplasms in mice. The Journal of Experimental Medicine, 211(3), 457-472.

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RNA Purification and Depletion Protocol

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The RNA aliquot was treated with Turbo DNase (Ambion) at 37°C for 30 to 60 min to eliminate DNA contamination. The resultant RNA was then used as a template for the PCR amplification of a housekeeping gene, rpoA (see Table S7 for primers); a negative PCR result provided evidence that the RNA was free of DNA contamination. PCR cycling conditions were as follows: 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Subsequently, the contaminating host RNA was depleted from the sample using a MICROBEnrich kit (Ambion) in accordance with the manufacturer’s instructions. A total of 5 μg of pooled RNA was subjected to repeated cycles of bacterial RNA enrichment to ensure the almost complete depletion of the host RNA. Next, bacterial rRNA was depleted from the preparation using a Ribo-Zero rRNA removal kit (Epicentre) per the manufacturer’s instructions. The quality and quantity of RNA obtained after the bacterial RNA enrichment and rRNA depletion were determined using a 2100 Bioanalyzer.

Low L.Y., Harrison P.F., Gould J., Powell D.R., Choo J.M., Forster S.C., Chapman R., Gearing L.J., Cheung J.K., Hertzog P, & Rood J.I. (2018). Concurrent Host-Pathogen Transcriptional Responses in a Clostridium perfringens Murine Myonecrosis Infection. mBio, 9(2), e00473-18.

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Isolation and Analysis of Splenic RNA

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Spleens were harvested at day 3 PI, homogenized, and RNA was isolated using the RNeasy kit (QIAGEN). Bacterial RNA was enriched using the MICROBEnrich kit (Ambion), and mouse tissue transcripts were detected from total RNA samples, prior to enrichment. qRT-PCR was performed as described above. See also Supplemental Experimental Procedures.

Davis K.M., Mohammadi S, & Isberg R.R. (2014). Community behavior and spatial regulation within a bacterial microcolony in deep tissue sites serves to protect against host attack. Cell host & microbe, 17(1), 21-31.

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