RNA profile diversity across arthropoda: guidelines, methodological artifacts, and expected outcomes

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• High-quality RNA is crucial for RNA sequencing (RNAseq) and downstream analyses.
• The RNA Integrity Number (RIN) is the standard metric for RNA quality but was developed using model organisms.
• Invertebrate 28S ribosomal RNA (rRNA) is susceptible to gap deletion, a denaturation that can be misinterpreted as degradation.

Purpose of the Study:

• To investigate the prevalence of 28S rRNA gap deletion in arthropods.
• To assess the limitations of current RNA quality metrics like RIN in nonmodel invertebrates.
• To determine if 28S rRNA collapse is a general pattern or artifact in arthropods.

Main Methods:

• Sampling across major arthropod lineages.
• Analysis of rRNA profiles using gel electrophoresis.
• High-throughput RNA sequencing and transcriptome assembly.
• Investigating the temperature-dependent nature of 28S denaturation.

Main Results:

• 28S rRNA collapse was observed in approximately 90% of sampled arthropod species.
• RNA profiles showed considerable diversity despite the prevalence of 28S collapse.
• High-quality transcriptomes were successfully generated from arthropods with collapsed 28S rRNA.
• 28S rRNA denaturation can occur at relatively low temperatures.

Conclusions:

• The phenomenon of 28S rRNA gap deletion is widespread in arthropods and often misinterpreted.
• Current RNA quality metrics (RIN) are inadequate for accurately assessing RNA quality in many nonmodel invertebrates.
• This study highlights the need for revised RNA quality assessment methods for diverse invertebrate taxa.