A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage...

Area of Science:

• Parasitology
• Genomics
• Molecular Biology

Background:

• Advancements in sequencing technology enable transcriptomics for malaria research.
• A comprehensive, up-to-date reference protocol for Plasmodium transcriptome generation is lacking.
• This study addresses the need for efficient high-throughput transcriptome data generation from Plasmodium-infected samples.

Purpose of the Study:

• To present a comprehensive methodology for generating full-length mRNA transcriptomes from Plasmodium-infected blood.
• To streamline the process for high-throughput studies.
• To optimize existing methods for Plasmodium RNA analysis.

Main Methods:

• Evaluated RNA-preserving reagents and storage conditions.
• Compared RNA extraction protocols to identify the most suitable method for low-volume, low-parasitaemia samples.
• Optimized the SMART-seq2 amplification for AT-rich Plasmodium falciparum RNA.
• Developed a modified CRISPR-Cas9 method for human hemoglobin transcript depletion.
• Applied the protocol to generate high-resolution transcriptomes for laboratory and field strains.

Main Results:

• Established an optimal method for high-quality total RNA extraction from challenging samples.
• Achieved high-quality transcriptomes from 10 ng total RNA and 0.05% parasitaemia.
• Improved parasite transcriptome coverage through targeted depletion of human transcripts.
• Generated the highest resolution transcriptome of the P. falciparum intraerythrocytic cycle to date.
• Created the largest transcriptome dataset from Southeast Asian field isolates.

Conclusions:

• The presented methodology provides an inclusive pipeline for generating quality transcriptomic data from diverse Plasmodium samples.
• The pipeline is adaptable for robotic handling, facilitating both small and large-scale malaria research.
• This work supports future transcriptomic studies in malaria pathology and epidemiology.