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Published on: November 13, 2014
Effects of PCR inhibitors on mRNA expression for human blood identification
Shusaku Matsumura1, Aya Matsusue2, Brian Waters2
1Forensic Science Laboratory, Fukuoka Prefectural Police Headquarters, 7-7 Higashikoen, Hakata-ku, Fukuoka 812-8576, Japan; Department of Forensic Medicine, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
Abstract:
Detection of body fluid-specific mRNAs, such as those specific for blood, using real-time polymerase chain reaction (PCR) has become a useful tool in forensic science. Blood stains often contain PCR inhibitors that may be co-extracted with RNA and adversely affect PCR. The effects of inhibitors on the detection of mRNA markers for blood identification, namely, hemoglobin beta (HBB) and actin beta, were examined herein. Inhibitors were added to a real-time PCR mix, reverse transcription mix, and blood samples before RNA extraction, and the following parameters: Ct, delta Ct (dCt), and melting temperature (Tm) values, were monitored. Hematin, humic acid, indigo carmine, and tannic acid were used as PCR inhibitors. The results showed that Ct values for HBB in samples containing inhibitors in their real-time PCR mix increased in a concentration-dependent manner, and were undetectable at higher concentrations. Moreover, Ct values for HBB in tannic acid-spiked samples reached a maximum once, and inhibition decreased at higher concentrations. dCt values increased in hematin-spiked samples, but decreased in tannic acid-spiked samples. Tm values decreased following the addition of each inhibitor. The reverse transcription reaction was scarcely inhibited at concentrations that markedly affected real-time PCR. The complete removal of inhibitors added to blood was difficult. However, the observed inhibitory effects were weaker than those when inhibitors were added to the PCR cocktail. PCR inhibition was effectively reduced by repurification of complimentary DNA with DNA extraction kits. These results will assist examiners in deducing contaminating inhibitors and selecting an appropriate method to remove them.
Insights
Forensic real-time PCR for blood identification can be hindered by PCR inhibitors. Repurifying complementary DNA effectively reduces inhibition, aiding forensic analysis.
Area of Science:
- Forensic Science
- Molecular Biology
- Biochemistry
Background:
- Real-time polymerase chain reaction (PCR) is crucial for detecting body fluid-specific mRNAs in forensic science.
- Blood stains frequently contain PCR inhibitors that can interfere with RNA extraction and subsequent PCR analysis.
- The impact of these inhibitors on mRNA markers like hemoglobin beta (HBB) and actin beta requires thorough investigation.
Purpose of the Study:
- To investigate the effects of common PCR inhibitors on the detection of blood-specific mRNA markers using real-time PCR.
- To evaluate the influence of inhibitors added at different stages (PCR mix, reverse transcription, and pre-extraction) on PCR parameters.
- To assess the efficacy of DNA repurification in mitigating PCR inhibition.
Main Methods:
- Real-time PCR was used to detect HBB and actin beta mRNA in blood samples.
- Common PCR inhibitors (hematin, humic acid, indigo carmine, tannic acid) were added to PCR mixes, reverse transcription mixes, and blood samples prior to RNA extraction.
- Key parameters monitored included Ct, delta Ct (dCt), and melting temperature (Tm) values.
- Complementary DNA repurification using DNA extraction kits was employed to reduce inhibition.
Main Results:
- Inhibitors increased Ct values in a concentration-dependent manner, leading to undetectable HBB at high concentrations.
- Tannic acid showed a unique inhibition pattern, with decreased inhibition at higher concentrations.
- Delta Ct values varied depending on the inhibitor and sample treatment.
- Melting temperature (Tm) values decreased upon inhibitor addition.
- Reverse transcription was minimally affected, while pre-extraction inhibitor addition showed weaker effects than direct PCR mix addition.
- Repurification of complementary DNA significantly reduced PCR inhibition.
Conclusions:
- PCR inhibitors significantly impact the detection of blood-specific mRNA markers, affecting Ct, dCt, and Tm values.
- The stage of inhibitor addition and the type of inhibitor influence the observed inhibitory effects.
- Repurification of complementary DNA is an effective strategy to mitigate PCR inhibition in forensic samples.
- Understanding inhibitor effects aids forensic examiners in selecting appropriate removal methods for accurate blood identification.
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