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September 10, 2025Open Access

Increasing casework and research congruency for RNA-based assays using two different transcripts from Chrysomya megacephala (Diptera: Calliphoridae)

Key Points

Successful gene expression analysis is critical for accurate larval age estimation and highlighting casework discrepancies.
After boiling and storage, 29 out of 30 larvae produced viable transformer transcripts, indicating strong RNA retention.
The research explores handling practices for chloroform-euthanized larvae compared to casework standards, aiming for methodological alignment.
Findings may enable better integration of gene expression techniques in forensic entomology, improving evidence reliability.

Abstract

One aspect to improve the operational utility of forensic science research is congruency in sample handling techniques between research and casework. For the field of forensic entomology, a common form of evidence is insect larvae removed from a body. Age estimation of the larvae is based on an attribute, commonly larval length, which is compared to a known reference growth curve for that species under similar environmental conditions. The casework recommendation is that larvae are hot water killed and stored in ethanol prior to size measurement. This handling approach is counterintuitive to methodology aimed at maintaining RNA integrity in gene expression research, an additional approach that could augment information gained from larval evidence and possibly increase precision in larval age estimation. In this study, the ability to obtain two different gene transcripts, transformer and rp49, after boiling and storing larvae, notably in ethanol, was evaluated using the forensically important blow fly Chrysomya megacephala. Success rates of obtaining sex-specific transcripts of the transformer gene and detection of rp49 across four different combinations of either live or boiled larvae stored in either RNAlater™ or ethanol were obtained after 2-3 weeks and 8-9 weeks storage at ~4°C. Across a total of 90 samples ranging in length from 3.32mm-15.84mm, only 3 larvae did not produce a sex-specific transformer PCR product and only 2 larvae did not produce an rp49 transcript. Specifically in relation to boiling and ethanol, all but 1 out of 30 larvae were successful for sex-specific transformer transcript detection, and all larvae were successful for rp49. These results suggest that approaches for larval handling in gene expression work should be re-evaluated to allow for conditions that better reflect casework practices.

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