This work was supported by NIH grant 1SC2GM092789-01A1, and MS was a research scholar of NMSU Howard Hughes Medical Institution Undergraduate Research Programs. The video was directed and produced by Amy Lanasa and coordinated by Dr. Philip Lewis with the Creative Media Institute at NMSU.

Procedure
1. Mosquito Rearing
<ol>
 <li>Rear mosquito Anopheles gambiae G3 strain in an insectary at 27.5 &deg;C with 80% humidity and 12:12 hr cycle of light/dark. </li>
 <li>Feed larvae with ground cat food with brewer's yeast at ratio 1:1. </li>
 <li>Feed adult mosquitoes on mice blood at day 3 post-emergence for egg production. </li>
</ol>
2. Mosquito Gut Dissection 
<ol>
 <li>Autoclave dissection tools (slides and forceps). </li>
 <li>Collect...

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat. Rev. Genet. 10, 57-63 (2009).</li><li>Xie, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing+the+Bemisia+tabaci+Transcriptome+Reveals+a+Highly+Diverse+Bacterial+Community+and+a+Robust+System+for+Insecticide+Resistance.">Pyrosequencing the Bemisia tabaci Transcriptome Reveals a Highly Diverse Bacterial Community and a Robust System for Insecticide Resistance.</a> PLoS One. 7, e35181 (2012).</li><li>Xie, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+metatranscriptome+of+the+protistan+community+in+Coptotermes+formosanus+with+emphasis+on+the+lignocellulolytic+system.">Profiling the metatranscriptome of the protistan community in Coptotermes formosanus with emphasis on the lignocellulolytic system.</a> Genomics. 99, 246-255 (2012).</li><li>Gosalbes, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metatranscriptomic+approach+to+analyze+the+functional+human+gut+microbiota.">Metatranscriptomic approach to analyze the functional human gut microbiota.</a> PLoS One. 6, e17447 (2011).</li><li>Urich, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+assessment+of+soil+microbial+community+structure+and+function+through+analysis+of+the+meta-transcriptome.">Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome.</a> PLoS One. 3, e2527 (2008).</li><li>Gilbert, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+large+numbers+of+novel+sequences+in+the+metatranscriptomes+of+complex+marine+microbial+communities.">Detection of large numbers of novel sequences in the metatranscriptomes of complex marine microbial communities.</a> PLoS One. 3, e3042 (2008).</li><li>Gifford, S. M., Sharma, S., Rinta-Kanto, J. M., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+a+deeply+sequenced+marine+microbial+metatranscriptome.">Quantitative analysis of a deeply sequenced marine microbial metatranscriptome.</a> ISME J. 5, 461-472 (2011).</li><li>Poretsky, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+day/night+metatranscriptomic+analysis+of+microbial+communities+in+the+North+Pacific+subtropical+gyre.">Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre.</a> Environmental microbiology. 11, 1358-1375 (2009).</li><li>Shrestha, P. M., Kube, M., Reinhardt, R., Liesack, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+activity+of+paddy+soil+bacterial+communities.">Transcriptional activity of paddy soil bacterial communities.</a> Environmental Microbiology. 11, 960-970 (2009).</li><li>Mettel, C., Kim, Y., Shrestha, P. M., Liesack, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+mRNA+from+soil.">Extraction of mRNA from soil.</a> Applied and Environmental Microbiology. 76, 5995-6000 (2010).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+two+ribosomal+RNA+removal+methods+for+microbial+metatranscriptomics.">Validation of two ribosomal RNA removal methods for microbial metatranscriptomics.</a> Nat. Meth. 7, 807-812 (2010).</li><li>Pang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+mRNA+purification+by+magnetic+capture-hybridization+method.">Bacterial mRNA purification by magnetic capture-hybridization method.</a> Microbiol. Immunol. 48, 91-96 (2004).</li><li>Stewart, F. J., Ottesen, E. A., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+quantitative+analyses+of+a+universal+rRNA-subtraction+protocol+for+microbial+metatranscriptomics.">Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics.</a> ISME J. 4, 896-907 (2010).</li><li>Wang, Y., Gilbreath, T. M., Kukutla, P., Yan, G., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+gut+microbiome+across+life+history+of+the+malaria+mosquito+Anopheles+gambiae+in+Kenya.">Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya.</a> PLoS One. 6, e24767 (2011).</li><li>Su, C., Sordillo, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+enrich+mRNA+from+total+prokaryotic+RNA.">A simple method to enrich mRNA from total prokaryotic RNA.</a> Mol. Biotechnol. 10, 83-85 (1998).</li><li>Gusmao, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture-dependent+and+culture-independent+characterization+of+microorganisms+associated+with+Aedes+aegypti+(Diptera:+Culicidae)+(L.)+and+dynamics+of+bacterial+colonization+in+the+midgut.">Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut.</a> Acta Trop. 115, 275-281 (2010).</li><li>Lindh, J. M., Terenius, O., Faye, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=16S+rRNA+gene-based+identification+of+midgut+bacteria+from+field-caught+Anopheles+gambiae+sensu+lato+and+A.+funestus+mosquitoes+reveals+new+species+related+to+known+insect+symbionts.">16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts.</a> Appl. Environ. Microbiol. 71, 7217-7223 (2005).</li><li>Simon, C., Daniel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+analyses:+past+and+future+trends.">Metagenomic analyses: past and future trends.</a> Appl. Environ. Microbiol. 77, 1153-1161 (2011).</li><li>Wang, Y., Qian, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservative+fragments+in+bacterial+16S+rRNA+genes+and+primer+design+for+16S+ribosomal+DNA+amplicons+in+metagenomic+studies.">Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.</a> PLoS One. 4, e7401 (2009).</li><li>Hunt, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+23S+rRNA+PCR+primers+for+use+in+phylogenetic+studies+of+bacterial+diversity.">Evaluation of 23S rRNA PCR primers for use in phylogenetic studies of bacterial diversity.</a> Appl. Environ. Microbiol. 72, 2221-2225 (2006).</li></ol>

The authors declare that they have no competing financial interests.

A complex microbial community resides in the mosquito gut ecosystem 14,16,17. Metatranscriptomic sequencing (RNA-seq) can reveal context dependent functional information by interrogating the entire microbial transcriptome4,18. Technically, oligo-d(T) mediated enrichment of prokaryotic mRNA is not applicable due to the absence of stable poly (A) tails of the messengers. Alternatively, rRNA depletion has been used for mRNA enrichment. Here, we developed a subtraction-based protocol to deplete rRNA usi...

Next-generation sequencing technology has greatly advanced metagenomics study by allowing to assess the taxonomic composition and genetic functionality of a microbial assemblage. RNA-Sequencing (RNA-Seq)1 can bypass culture-based methods to investigate microbial metatranscriptomes in different contexts 2-5. A major obstacle to microbial RNA-seq is the difficulty in enriching mRNA, as the prokaryotic mRNA species are not stably polyadenylated. Therefore, oligo d(T) mediated messenger enrichment is not applicable. Removal of abundant rRNA is an alternative approach to enrich mRNA. Commercial rRNA depletion kits, such as Microexpress Bacterial mRNA Enrichment kit (Ambion), RiboMinus Transcriptome Isolation Kit (Bacteria) (Life Technologies), and mRNA-ONLY Prokaryotic mRNA Isolation kit (Epicentre) that preferentially degrades rRNA with an exonuclease, have been used for removing rRNA 6-8. However, the capture probes in Microexpress or RiboMinus are good for removing known rRNA from typical Gram-positive and Gram-negative bacteria (see manufacturers' specifications), but less compatible with rRNA from unknown microbes. Consequently, the removal may be less efficient for metagenomic samples8-10. Besides, the mRNA abundance fidelity was questionable when the exonuclease treatment was applied 11. Overall, subtraction-based rRNA depletion was less biased and more effective in mRNA enrichment in metagenomic settings 10-13. 
The mosquito gut accommodates a dynamic microbial community14. We are interested in characterizing the function of the mosquito gut microbiome by using RNA-Seq. In an RNA sample isolated from the mosquito guts, both mosquito and microbial RNA are present. Here, we describe a modified protocol to use community specific rRNA probes to efficiently deplete mosquito and microbial rRNA by subtractive hybridization. The resultant mRNA enriched samples are appropriate for RNA-Seq. The overall workflow is depicted in Figure 1.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material </td>
 </tr>
 <tr>
 <td>Meta-G-Nome 				DNA isolation kit</td>
 <td>Epicentre</td>
 <td>MGN0910</td>
 <td>Metagenomic 				DNA isolation</td>
 </tr>
 <tr>
 <td>TriPure </td>
 <td>Roche</td>
 <td>11667165001</td>
 <td>Mdetagenomic 				RNA isolation</td>
 </tr>
 <tr>
 <td>MEGAscript 				T7 kit</td>
 <td>Ambion</td>
 <td>AM1334</td>
 <td>In 				vitro synthesis of RNA probes</td>
 </tr>
 <tr>
 <td>RNaseZap</td>
 <td>Ambion</td>
 <td>AM9780</td>
 <td>RNase 				free working area </td>
 </tr>
 <tr>
 <td>Biotin-16-UTP</td>
 <td>Roche</td>
 <td>11388908910</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Biotin-11-CTP</td>
 <td>Roche</td>
 <td>4739205001</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Streptavidin 				magnetic beads</td>
 <td>NEB</td>
 <td>S1420S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>Magnetic 				separation rack</td>
 <td>NEB</td>
 <td>S1506S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>RNeasy 				mini kit</td>
 <td>QIAGEN</td>
 <td>74104</td>
 <td>Purification 				of subtracted RNA</td>
 </tr>
 <tr>
 <td>RNase-Free 				DNase Set</td>
 <td>QIAGEN</td>
 <td>79254</td>
 <td>Removal 				DNA contamination </td>
 </tr>
 <tr>
 <td>Agilent 				RNA 6000 Pico Kit</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>5067-1513</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Bio-Gen 				PRO200 				Homogenizer</td>
 <td>PRO 				Scientific</td>
 <td>01-01200</td>
 <td>Mosquito 				gut tissue homogenization</td>
 </tr>
 <tr>
 <td>NanoDrop 				1000 Spectrophotometer</td>
 <td>Thermo 				Scientific</td>
 <td></td>
 <td>DNA 				&amp; RNA quantitation</td>
 </tr>
 <tr>
 <td>2100 				Bioanalyzer</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>G2940CA</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 </tbody>
</table>

depletion of ribosomal rna for mosquito gut metagenomic rna-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

The protocol includes three sections: (1) preparation of metagenomic DNA templates for rRNA PCR; (2) creation of sample specific capture rRNA probes; (3) depletion of rRNA from total RNA by subtractive hybridization. Isolation of high quality metagenomic DNA and RNA is essential to the entire process. The modified Meta-G-Nome DNA isolation protocol yields high-quality metagenomic DNA from mosquito guts, as shown in Figure 2. It can be challenging to isolate high-quality total RNA from mosquito guts. This...

Watch this Scientific Journal Video about Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq at JoVE.com

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

depletion-of-ribosomal-rna-for-mosquito-gut-metagenomic-rna-seq

Research

JoVE Journal

Biology

19.5K Görüntüleme.  New Mexico State University. A ribozomal RNA (rRNA) azalması protokolü sivrisinek bağırsak metatranscriptome RNA-seq mesajcı RNA (mRNA) zenginleştirmek için geliştirilmiştir. Çıkarma yoluyla rRNA kaldırmak için kullanılan örnek belirli rRNA probları, sivrisinek ve onun bağırsak mikropları oluşturulmuş. Protokol performans rRNA yaklaşık olarak% 90-99 arasında kaldırma neden olabilir.