Journal of Forensic Science and Medicine

BRIEF COMMUNICATION
Year
: 2022  |  Volume : 8  |  Issue : 2  |  Page : 76--79

The impact of different stain carriers on the mrna profiling from bloodstains


Hemiao Zhao1, Qingluan Lin2, Qi Zhang3, Jing Chen1, Zheng Tu1, Ruiqin Yang3, Lan Hu1, Chong Wang1,  
1 Division of Forensic Genetics, Key Laboratory of Forensic Genetics, Institute of Forensic Science, Ministry of Public Security, Beijing, China
2 Qionghai Police Bureau, Hainan Province, China
3 School of Crime Investigation, Chinese People's Public Security University, Beijing, China

Correspondence Address:
Chong Wang
Key Laboratory of Forensic Genetics, Institute of Forensic Science, Ministry of Public Security, Beijing 100 038
China
Lan Hu
Key Laboratory of Forensic Genetics, Institute of Forensic Science, Ministry of Public Security, Beijing 100 038
China

Abstract

Unlike DNA profiling, mRNA profiling is greatly affected by external factors. To analyze the influence of different stain carriers on the detectability of mRNA markers from bloodstains, this study examined 10 carriers, including a knife, cotton swab, paper, plastic, leather, cement, chopsticks, clothes, ceramic block, and wall. After detecting five specific mRNA markers (HBA, HBB, ALAS2, GYPA, and SPTB) and the housekeeping gene B2M in peripheral blood samples, no statistically significant differences in the effects of the carriers were found. The results suggest that when performing mRNA testing on bloodstains, the effect of the stain carrier has little influence.



How to cite this article:
Zhao H, Lin Q, Zhang Q, Chen J, Tu Z, Yang R, Hu L, Wang C. The impact of different stain carriers on the mrna profiling from bloodstains.J Forensic Sci Med 2022;8:76-79


How to cite this URL:
Zhao H, Lin Q, Zhang Q, Chen J, Tu Z, Yang R, Hu L, Wang C. The impact of different stain carriers on the mrna profiling from bloodstains. J Forensic Sci Med [serial online] 2022 [cited 2022 Nov 26 ];8:76-79
Available from: https://www.jfsmonline.com/text.asp?2022/8/2/76/348651


Full Text



 Introduction



The detection and analysis of bloodstains have always been important parts of forensic casework. DNA testing, the main detection method, provides significant data and information for individual identification and criminal suspect identification. However, considering the complex criminal environment, it is difficult to meet the investigation requirements only using the information provided by conventional DNA tests. With the development of new technologies, RNA profiling methods have become more widely used in the forensic medicine research field. These techniques are applied to estimate the time of death, infer the time of wound or plaque formation, and determine the type of body fluid.[1],[2],[3] At a crime scene, bloodstains can be deposited on different objects or surfaces such as various tools, ground, clothes, and walls. The impact of these various carriers on RNA detection from bloodstains has been infrequently studied.[4] In this study, we classified the carriers commonly found in cases into permeable, semipermeable, and nonpermeable objects. We also analyzed the RNA profiling results from different carriers to examine their impact on RNA detection.

 Materials and Methods



Chemicals and materials

QIAamp RNA Blood Mini Kit (Qiagen, Germany), SuperScript Ⅲ First-Strand cDNA Synthesis Kit (Invitrogen, USA), Mag Attract M48 DNA Manual Kit (Qiagen), HotStar Taq DNA Polymerase Kit (Qiagen), Typer19 Kit, Internal standard and Ladder (Institute of Forensic Science, Ministry of Public Security, China), Hi-Di Formamide (Applied Biosystems, USA), β-mercaptoethanol (chemically pure) (Amresco, USA), and absolute ethanol (Analytical pure, Sinopharm Medicine Group Chemical Reagent Company, China) were used.

Sample preparation

In this study, 10 fresh blood samples were collected from healthy volunteers, which included five men and five women ranging in age from 20 to 60 years old. Blood samples were collected by venipuncture into tubes containing an anticoagulant and stored at-20°C after collection.

The carriers included in this study are commonly found in forensic casework. They were grouped by the permeability of the blood: permeable objects, semipermeable objects, and nonpermeable objects. Permeable carriers: cotton swab group (marked as MQ group), clothing group represented by a cotton T-shirt (marked as YF group); semipermeable carriers: wooden material group represented by chopsticks (marked as KZ group), paper material group (marked as NP group) represented by kraft paper, wall surface group (marked as QB group) represented by white lime flakes, cement floor group (marked as SN group); and nonpermeable carriers: leather material group (marked as PG group), ceramic tile group (marked as CZ group), and plastic carrier represented by a black plastic bag (marked as SL group), metal group represented by a knife (marked as DP group). For this research, 20 cotton swabs, 20 round cotton sheets (diameter 5 cm), 20 wooden disposable chopsticks, 20 pieces of round kraft paper (diameter 5 cm), 20 lime slices (diameter 5 cm), cement floor, 20 pieces of leather, 20 ceramic tiles blocks, 20 black plastic bags, and 20 pocket knives were collected.

Then, 50 μL of blood from each sample was separately spread on the above carriers and allowed to air dry. Each sample was divided into Groups A and B and tested in parallel. All samples were stored in a dry and ventilated room until RNA extraction.

RNA preparation

Total RNA was isolated using a QIAamp RNA Blood Mini Kit (Qiagen), and then reverse transcription was carried out with a SuperScript Ⅲ First-Strand cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions.

The RNA markers were acquired from previous reports and Typer19 kit fluorescence verification. The sequences are shown in [Table 1].{Table 1}

A 25 μL amplification system was set up to carry out endpoint polymerase chain reaction (PCR), performed with the reagents shown in [Table 2].{Table 2}

Capillary electrophoresis and profile analysis

Capillary electrophoresis was run on a 3500 × L Genetic Analyzer (Applied Biosystems) under the following conditions: 1 μL PCR product combined with 10 μL Typer500 interior label premixed with Hi-Di Formamide. The electrophoresis data genotyping was performed on GeneMapper ID-X (Applied Biosystems) 1.4 and analyzed using the SPSS13.0 (IBM).

 Results



Identification of blood markers

One housekeeping gene (B2M) and five specific messenger RNA (mRNA) markers (HBA, HBB, ALAS2, SPTB, and GYPA) were detected in this experiment. The number of amplifications (total of 20) and the average relative fluorescence units (RFUs) are shown in [Table 3] and [Table 4].{Table 3}{Table 4}

The R × C contingency table was used for Chi-square tests. The 0% expected number was <5 and the Chi-square value was 61.095 (P > 0.05), indicating that there was no statistically significant difference in the number of marker amplifications on each carrier. To meticulously analyze each carrier's effect on marker detection, RFU values were recorded and evaluated.

Carrier comparison

The RFU values of each marker were analyzed as the dependent variable by one-way analysis of variance, and any differences between carriers were calculated. The results are shown in [Table 5].{Table 5}

The above results suggest that each marker has different RFU values for the various carriers. To clarify the differences between the carriers, we conducted a Student‒Newman‒Keuls ex-post analysis. The tests showed that when setting the α-level to 0.05, the RFU values of the six mRNA markers can be divided into at least two groups (HBA, GYPA) and at most six groups (SPTB).

For the housekeeping gene B2M, the RFU values of four carriers, blade, cement, chopsticks, and clothing, were significantly lower than those of the other six carriers. In addition, the differences between the four carriers were statistically significant. For HBA, the RFU value of the kraft paper group was remarkably lower than that of the other nine carriers. For HBB, the chopsticks group had the lowest RFU value compared with the other seven carriers (the cement and leather groups had incorrect RFU values and no calculations were performed). The test results of ALAS2 could be divided into three groups: the RFU value of the knife group was the lowest, those of the clothes, kraft paper, and ceramic tile groups were moderate, and those of the remaining six carriers were high. The STPB group's results were more complicated and could be divided into six groups: the lowest RFU value was still from the knife group, whereas the RFU values of the cement, leather, and plastic groups were high. In the GYPA group, the RFU values of four carriers, leather, clothes, plastics, and chopsticks, were low (277–473), which were significantly different from the RFU values of the other six carriers (10,889–26,131).

 Discussion



Effect of carriers on mRNA marker detectability

The Chi-square analysis suggested that the ten bloodstained carriers involved in this study have no practical effect on the successful detection of the mRNA markers. Overall, the material differences between the carriers do not affect the detection of related mRNA markers from bloodstains. These findings imply that the influence of the carrier can be ignored in terms of marker detectability.

Effects of carriers on relative fluorescence unit values

Our analysis suggested that there were statistical differences between the RFU values of the mRNA markers on each carrier. Overall, the knife group had the lowest RFU values among the multiple markers (~4800 in SPTB group, ~8000 in ALASA group, etc.) and was therefore the least satisfactory for mRNA profiling. This is possible because the pocket knife is made of metal, which is prone to rusting. Contamination with such impurities can affect successful marker detection by leading to quicker degradation of the mRNAs. Four carriers–cotton swabs, ceramic blocks, walls, and kraft paper–had the best detection results. Their RFU values were >20,000. The remaining five carriers' results were moderate. In general, the results from the nonpermeable carriers (such as a knife) were slightly worse than those from the permeable carriers (such as cotton swabs). This may be related to the permeability of the carrier, as more permeability allows for greater absorption of blood into the internal structure. This increases the number of cells present and therefore a higher amount of RNA can be extracted. In addition, because this type of internal structure has a protective function, the mRNAs can be considerably less affected by external environmental factors.

Marker differences

The data in [Table 3] and [Table 4] demonstrate that among the five types of mRNA markers specific to peripheral blood, the detectable rate and RFU value of GYPA are both the lowest. Therefore, the selection should be performed carefully. The detectable rates and RFU values of HBA and HBB were high, making them ideal markers for peripheral blood testing. However, in practice, we found that because of the high sensitivity of HBA and HBB detection, any trace amounts of blood components in other body fluids may result in two positive results. This strong sensitivity also brings difficulties in testing. For example, when using second-generation sequencing for mRNA detection, highly sensitive signals may have a masking effect on other markers.

Issues to be noted in practice

The blood sample volume used in this study was 50 μL, which is similar to what is commonly seen in forensic casework. Our analysis suggested that most of the mRNA markers we tested can be successfully detected from bloodstains on carrier samples. Differences are only reflected in the RFU values. Therefore, we believe that storing test materials at ideal conditions will result in no obvious effects of the specific carrier on the detection of blood-related mRNA markers, suggesting that the carrier can be ignored in specific applications.

However, this study only involves a single storage condition and does not consider the impact of environmental changes on sample storage. In subsequent studies, the effects of environmental factors, such as temperature, humidity, and ultraviolet radiation, should be comprehensively analyzed.

 Conclusion



In this research, we examined the effects of 10 different carriers on the detection of certain mRNA markers from bloodstains. Our results suggest that the differences in carrier material did not affect mRNA marker detectability but did affect the specific RFU values. Therefore, under ideal test conditions in a laboratory, the effect of the carrier does not need to be considered for the detection of mRNA markers from bloodstains. If further quantitative analysis is required, then any difference between the carriers should be considered.

Ethics statement

The collection procedure was approved by the Human Ethics Committee of the Institute of Forensic Science, Ministry of Public Security (Approval No. 2020003, approved on March 15, 2020). This study adhered to the ethical principles of the Helsinki Declaration of the World Medical Association.

Acknowledgment

This study is funded by the basic research fund of the central public welfare research institute (2020JB001).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.[8]

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