High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation

Seiji Kubo; Keito Amai; Fumitaka Nakano; Jin Tanaka; Hideki Niimi

doi:10.1016/j.ab.2024.115727

High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation

Seiji Kubo^*, Keito Amai, Fumitaka Nakano, Jin Tanaka, Hideki Niimi^*

^*Corresponding author for this work

Clinical Laboratory and Molecular Pathology

Research output: Contribution to journal › Article › peer-review

Abstract

Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (RT-qPCR) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step RT-qPCR assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 μL of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for RT-qPCR. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.

Original language	English
Article number	115727
Journal	Analytical Biochemistry
Volume	698
DOIs	https://doi.org/10.1016/j.ab.2024.115727
State	Published - 2025/03

Keywords

PRM2
RT-qPCR
Semen
Sperm

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.ab.2024.115727

Cite this

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title = "High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation",

abstract = "Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (RT-qPCR) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step RT-qPCR assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 μL of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for RT-qPCR. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.",

keywords = "PRM2, RT-qPCR, Semen, Sperm",

author = "Seiji Kubo and Keito Amai and Fumitaka Nakano and Jin Tanaka and Hideki Niimi",

note = "Publisher Copyright: {\textcopyright} 2024 Elsevier Inc.",

year = "2025",

month = mar,

doi = "10.1016/j.ab.2024.115727",

language = "英語",

volume = "698",

journal = "Analytical Biochemistry",

issn = "0003-2697",

publisher = "Academic Press Inc.",

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TY - JOUR

T1 - High-throughput sperm screening using one-step RT-qPCR

T2 - Improvement and re-evaluation

AU - Kubo, Seiji

AU - Amai, Keito

AU - Nakano, Fumitaka

AU - Tanaka, Jin

AU - Niimi, Hideki

PY - 2025/3

Y1 - 2025/3

N2 - Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (RT-qPCR) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step RT-qPCR assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 μL of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for RT-qPCR. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.

AB - Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (RT-qPCR) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step RT-qPCR assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 μL of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for RT-qPCR. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.

KW - PRM2

KW - RT-qPCR

KW - Semen

KW - Sperm

UR - http://www.scopus.com/inward/record.url?scp=85211059768&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2024.115727

DO - 10.1016/j.ab.2024.115727

M3 - 学術論文

C2 - 39608625

AN - SCOPUS:85211059768

SN - 0003-2697

VL - 698

JO - Analytical Biochemistry

JF - Analytical Biochemistry

M1 - 115727

ER -

High-throughput sperm screening using one-step RT-qPCR: Improvement and re-evaluation

Abstract

Keywords

ASJC Scopus subject areas

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