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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Published: April 7th, 2013

DOI:

10.3791/50093

1Department of Biology, New Mexico State University

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 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.

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 ....

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Procedure

1. Mosquito Rearing

  1. Rear mosquito Anopheles gambiae G3 strain in an insectary at 27.5 °C with 80% humidity and 12:12 hr cycle of light/dark.
  2. Feed larvae with ground cat food with brewer's yeast at ratio 1:1.
  3. Feed adult mosquitoes on mice blood at day 3 post-emergence for egg production.

2. Mosquito Gut Dissection

  1. Autoclave dissection tools (slides and forceps).
  2. Collect.......

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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.......

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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.......

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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.

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

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