Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq
April 7th, 2013
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.
Hello Everyone. This is Fana Kla from the laboratory of Dr.Xenon Zu, department of Biology at New Mexico State University. We are working on the metagenomics of mosquito gut ecosystem.
Today we are going to demonstrate your A process to deplete ribosomal RNA from the total RNA samples to enrich messenger, RNA for RNA-Seq. RNA-Seq is a widely used technique to study the functions of microbial community. Firstly, I'm going to demonstrate to you how to isolate total RNA and metagenomic, DNA from muscular gut.
Analys Gambia mosquitoes are maintained at an insectory. Mosquitoes are first fed on sugar meal before, and then afterward female mosquitoes are fed on mice for ACHE production. These mosquitoes are fed on sugar and these are fed with mouse blood.
Prior to the procedure, all dissection tools and slides were sterilized. The mosquitoes are surface cleaned with 70%ethanol three times in a Petri dish, and placed back on a glass slide to dry out the ethanol, place the mosquitoes on a different glass slide and put a drop of sterile one XPBS solution. Then gently pull out the gut.
The mosquito guts will be used to get metagenomic DNA and total RNA respectively. Before we begin the RNA work, it is important to take standard precautions to avoid RNA degradation by maintaining a ribonuclease free environment while wearing gloves, clean the work area pipettes, and all required apparatuses with r nay sap. Prior to the procedure, collect 50 guts and place them in a 1.5 milliliter einor tube containing pu reagents.
For total RNA isolation genomic, DNA is isolated from another batch of 50 guts using a metagenomic isolation kit. The concentration of RNA or DNA is measured using NanoDrop. We used our RNA subtraction to remove ribosomal RNA from the total RNA sample.
First, the sample specific RRNA fragments are PCR amplified from the genomic DNA, which we took from the gut samples. The PCR products are purified using a Qiagen PCR purification kit. In this step, the T seven RNA polymerase is used to synthesize antisense RRNA probes.
Set up a reaction using the PCR product from step one and Biotinylated CTP and UTP. Then incubate the reaction overnight at 37 degrees Celsius. Now the antisense RRNA probes are ready to use for RRNA depletion.
This step employs BIOTINYLATED RRNA probes to catch target RRNA in the total RNA sample, the target RRNA and the RRNA probe. Hybrids are then captured by strep dividing coated magnetic beads. Set up the hybridization reaction in a PCR tube and incubate it in a thermal cycler for five minutes at 70 degrees Celsius.
Then ramp down the temperature to 25 degrees Celsius. To allow the RRNA hybridization while the reaction is running, prepare the beads. First, make three aliquots.
Using 100 microliter beads per tube. Wash the beads with one XSSC solution three times. Then leave the tubes on ice.
Once the reaction is ready, diluted by adding 100 microliter one XSSC with 20%form amide. Transfer the reaction solution to the tube with the beads. Mix well and then incubate at room temperature for 10 minutes.
Flicking the tube occasionally, spin the tube briefly and place it on the magnetic rack. Transfer the super natin to a collection tube. Wash the beads two more times each with 100 microliter.
One XSSC buffer solution added. Mix well and then recapture the beads on the magnetic rack. Transfer the supernatants to the collection tube.
Now we have an RRNA depleted RNA sample in the tube. Perform purification using the RN easy minikit from Qiagen. Following the instructions, now we are checking the effectiveness of the depletion.
The RNA samples are loaded onto an Agilent RNA 6, 000 PICO chip and run through the 2100 bioanalyzer. Before subtraction. There were two ribosomal RNA peaks 16 s and 23 s respectively.
After subtraction, the RRNA peaks were effectively removed. The remaining RNA is in the size range from 200 base pair to four kilobase. Now the RNA sample is ready to make a CD NA library for transcriptome sequencing.
We just walk you through the procedure to enrich the messenger, RNA for RNA.Seek. Thank you for watching us.
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Overview
This article presents a protocol for depleting ribosomal RNA (rRNA) to enrich messenger RNA (mRNA) from mosquito gut samples for RNA sequencing (RNA-seq). The method utilizes sample-specific rRNA probes to effectively remove rRNA, achieving a depletion rate of approximately 90-99%.
Key Study Components
Area of Science
- Metagenomics
- RNA sequencing
- Microbial ecology
Background
- Understanding the mosquito gut ecosystem is crucial for studying vector-borne diseases.
- RNA-seq is a powerful tool for analyzing the functions of microbial communities.
- Ribosomal RNA constitutes a significant portion of total RNA, complicating mRNA analysis.
- Effective rRNA depletion enhances the quality of RNA-seq data.
Purpose of Study
- To develop a reliable rRNA depletion protocol for mosquito gut metatranscriptome analysis.
- To improve the enrichment of mRNA for subsequent RNA-seq applications.
- To facilitate a better understanding of the microbial community in the mosquito gut.
Methods Used
- Isolation of total RNA and metagenomic DNA from mosquito gut samples.
- Design and application of sample-specific rRNA probes for rRNA depletion.
- Assessment of rRNA removal efficiency through quantitative analysis.
- Preparation of enriched mRNA for RNA-seq analysis.
Main Results
- The developed protocol successfully removed 90-99% of rRNA from total RNA samples.
- Enriched mRNA samples showed improved quality for RNA-seq.
- The method is applicable to various mosquito species and their associated gut microbes.
- Results contribute to a deeper understanding of the mosquito gut metatranscriptome.
Conclusions
- The rRNA depletion protocol is effective for enriching mRNA from mosquito gut samples.
- This advancement will enhance future studies on microbial communities in mosquitoes.
- Further research can build on these findings to explore ecological interactions in the gut.
Frequently Asked Questions
Executive Industry Relevance
Efficient depletion of ribosomal RNA from mosquito gut metagenomic samples enables high-fidelity transcriptomic profiling of complex microbial communities. This capability is critical for target validation and mechanistic de-risking in vector-borne disease research pipelines. The protocol supports predictive confidence in early discovery and translational studies by ensuring quantitative mRNA analysis from challenging biological matrices.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of microbial gene expression relevant to mosquito physiology and vector competence.
- Supports functional target validation by clarifying microbial contributions to host traits.
- Facilitates mechanistic de-risking through accurate transcriptomic readouts.
Screening & Assay Development
- Prepares rRNA-depleted RNA suitable for standardized RNA-seq workflows.
- Improves assay reproducibility by minimizing rRNA interference in downstream analyses.
- Enables quantitative assessment of microbial mRNA for reliable compound or intervention screening.
Translational & Preclinical Research
- Aligns metatranscriptomic data with disease-relevant models for translational biomarker discovery.
- Supports continuity from discovery through preclinical validation by enabling robust microbial gene expression profiling.
- Reduces biological ambiguity in preclinical decision-making for vector-targeted interventions.
Pipeline & Workflow Integration
This rRNA depletion protocol integrates at the interface of early discovery and lead identification, providing high-quality RNA inputs for transcriptomic and functional genomics workflows.
- Discovery Biology: Enhances hypothesis testing by enabling comprehensive microbial transcriptome analysis.
- Screening: Delivers rRNA-depleted RNA for reproducible, quantitative RNA-seq assays.
- Analytics: Provides clear mRNA readouts, supporting statistical comparison of experimental conditions.
- Translational Research: Facilitates biomarker alignment and mechanistic insights in disease-relevant mosquito models.
- Enterprise Reuse: Offers a reusable protocol adaptable to diverse metagenomic and host-microbe systems.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in microbial transcriptomics.
- Operational Value: Standardizes RNA preparation for scalable, reproducible RNA-seq workflows.
- Strategic Value: Improves go/no-go decisions by enabling robust target and pathway validation.
- Portfolio Impact: Supports risk-adjusted prioritization of vector-targeted discovery programs.
Implementation Considerations
- Requires expertise in RNA handling and metagenomic sample preparation.
- Needs access to PCR, in vitro transcription, and magnetic bead-based separation infrastructure.
- Demands cross-team standardization for probe design and depletion efficiency assessment.
- Adaptable to various mosquito species and microbial communities with protocol optimization.
- Effectiveness depends on probe specificity and careful RNA quality control.
Biopharma FAQs
Chapters in this video
0:02
Title
0:08
Introduction
0:48
Mosquito Rearing
1:31
Mosquito gut dissection
1:43
Metagenomic DNA and RNA isolation
2:27
Ribosomal RNA depletion
2:34
PCR amplification of rRNA gene fragments
2:48
In vitro transcription of rRNA probes
3:19
rRNA subtraction
3:39
Removal of rRNA
5:07
Representative results
5:48
Conclusion
