The Effect of Elapsed Time on the Quantity of mRNA in Skin: A Study to Evaluate the Potential Forensic Use of mRNA to Determine the Postmortem Interval

Hye Jong Song; So Young Kang; Jong Min Chae

doi:10.7580/KoreanJLegMed.2012.36.2.151

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Song, Kang, and Chae: The Effect of Elapsed Time on the Quantity of mRNA in Skin: A Study to Evaluate the Potential Forensic Use of mRNA to Determine the Postmortem Interval

Original Article

Korean J Leg Med 2012;36(2):151-158.

Published online: 10 December 2012

DOI: https://doi.org/10.7580/KoreanJLegMed.2012.36.2.151

The Effect of Elapsed Time on the Quantity of mRNA in Skin: A Study to Evaluate the Potential Forensic Use of mRNA to Determine the Postmortem Interval

Hye Jong Song¹, So Young Kang¹, Jong Min Chae²

¹Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

²Department of Forensic Medicine, Kyungpook National University School of Medicine, Daegu, Korea

Corresponding Author: Hye Jong Song Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Present Address: 1223 SW 5th Court, Fort Lauderdale, Florida, USA 33312 TEL: +1-305-748-5716 E-mail: songhyejong@gmail.com

Received 25 September 2012 Revised 21 October 2012 Accepted 22 November 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Ribonucleic acid (RNA) has potential use in forensic science for the determination of postmortem interval. We report the first study on serial sampling of messenger RNA (mRNA) from surgical specimens to determine if there is a correlation between mRNA quantity and elapsed time. Skin tissues were collected from modified radical mastectomy specimens. After a defined period of time, bisected skin sections were cut and frozen in liquid nitrogen. Serial collection of the specimens was conducted, and frozen sections were obtained from all samples. Quantitative real-time reverse transcription-polymerase chain reaction was performed using the extracted RNA to measure the transcriptional activity of 2 selected housekeeping genes. The selected loci were mRNA sequences that exhibited time-dependent quantitative changes in a previous study. We collected 44 samples from 9 different patients, with 3–10 samples collected per patient. The amount of mRNA transcripts present in the serial samples showed a weak time-dependent correlation trend only in some cases. Further studies to evaluate different target mRNA sequences are necessary, as is exploration of additional methods to evaluate mRNA transcript degradation.

Keywords: messenger RNA, postmortem interval, reverse transcription-polymerase chain reaction

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Fig. 1.

The schematic shows the locations and size of target sequences on each mRNA.

Fig. 2.

(A) This image shows a representative presentation of electrophoresis of extracted RNA compared to positive control (PC) with visible band for 18S (lower arrow) and 28S (upper arrow). (B) This plot shows the amplification of sequences in realtime PCR.

Fig. 3.

These graphs show the relationship between the Ct values with elapsed time.

Fig. 4.

These graphs show the relative quantifications of FASN1 along elapsed time (calibrator: FASN2).

Fig. 5.

These graphs show relative quantifications of FASN1 along elapsed time (calibrator: ACTB1).

Table1.

Specification of the Genes Tested in the Present Study

Symbol	Name	Function	Localization
ACTB	Actin, beta	Cytoskeletal structural protein	07p22
FASN	Fatty acid synthase	Enzyme for long-chain saturated fatty acids synthesis	17q25

Table 2.

Primer and Probe Sequences

Sequence Name	Primer Sequence	Probe Sequence	Size
FASN1^*	5’-CTCATCCGCTCGTTGTACCAG-3’	FAM^�-CCGGAGTGGCCCCT-NFQ	300bp
	5’-GCTGGGATCTCAGGGTTGG-3’
FASN2	5’-GGTCTTGAGAGATGGCTTGCTG-3’	FAM-TTCCAGGACGTCTGCA-NFQ	195bp
	5’-TTGGCAAAGCCGTAGTTGCT-3’
ACTB1^�	5’-CAGATCATGTTTGAGACCTTCAACA-3’	FAM-TCCTCACCGAGCGCG-NFQ	346bp
	5’-GTGGCCATCTCTTGCTCGAA-3’
ACTB2	5’-ATCCACGAAACTACCTTCAACTCC-3’	FAM-TGGCATTGCCGACAGG-NFQ	177bp
	5’-AAGGAGCAATGATCTTGATCTTCAT-3’

^* FASN, Fatty acid synthase;

^� ACTB, Actin, beta;

^� FAM, Fluorescein amidite

Table 3.

Ct Values of each RNA Segment and Relative Quantification by the C_t Method

Case	Serial Number	Elapsed Time (h)	ΔCt				2^-ΔΔ Ct (Target/Reference)
Case	Serial Number	Elapsed Time (h)	FASN1	FASN2	ACTB1	ACTB2	FASN1 /FASN2	FASN1 /ACTB1	ACTB2/ ACRB1
1	1	18.0	33.31	25.57	20.96	26.50	01.00	001.00	1.00
	2	21.0	33.42	26.46	21.48	27.01	01.72	001.33	1.00
	3	39.0	33.12	27.25	21.83	27.27	03.66	002.09	1.07
	4	42.0	35.38	28.06	22.44	27.91	01.33	000.67	1.06
	5	45.0	34.21	28.04	22.46	27.95	02.98	001.52	1.03
	6	48.5	35.75	28.48	22.95	28.73	01.38	000.73	0.85
	7	63.0	35.83	28.37	23.16	28.83	01.21	000.80	0.91
	8	66.0	36.79	27.91	22.46	27.92	00.45	000.25	1.06
	9	68.0	34.07	27.85	22.31	28.27	02.86	001.50	0.74
2	1	13.0	38.00	27.58	21.75	27.52	01.00	001.00	1.00
	2	16.0	34.60	27.47	22.20	27.08	09.76	014.40	1.86
	3	19.0	33.34	27.79	21.90	26.71	28.94	027.81	1.94
	4	22.0	33.27	27.94	21.98	26.39	34.00	031.05	2.56
	5	37.0	36.08	28.64	23.21	27.90	07.85	010.39	2.12
	6	40.0	31.63	27.77	22.79	27.15	93.72	169.29	2.65
3	1	14.5	31.63	27.99	23.47	28.30	01.00	001.00	1.00
	2	17.5	33.35	27.49	23.01	27.46	00.22	000.22	1.30
	3	20.3	33.74	28.07	23.12	27.27	00.25	000.18	1.61
	4	24.0	33.19	27.57	22.52	26.53	00.25	000.18	1.77
	5	41.5	35.38	28.03	22.92	27.15	00.08	000.05	1.52
4	1	16.5	33.38	28.68	23.51	28.00	01.00	001.00	1.00
	2	19.5	34.19	30.49	25.33	30.02	02.01	002.01	0.87
	3	21.5	37.97	30.00	24.50	29.00	00.10	000.08	0.99
	4	24.5	37.01	29.36	23.96	28.23	00.13	000.11	1.17
5	1	17.5	34.06	27.38	21.74	25.53	01.00	001.00	1.00
	2	19.0	34.62	27.11	21.50	25.34	00.56	000.57	0.97
	3	21.0	33.02	27.03	21.49	25.87	01.61	001.72	0.66
	4	24.0	32.38	27.20	21.55	25.87	02.82	002.80	0.69
	5	46.0	34.28	27.46	21.74	25.98	00.91	000.86	0.73
6	1	00.0	33.91	26.71	20.86	24.48	01.00	001.00	1.00
	2	02.0	33.02	26.95	21.18	25.02	02.19	002.31	0.86
	3	05.0	29.89	26.13	19.11	22.47	10.81	004.83	1.20
	4	27.0	30.95	27.60	21.60	35.03	14.41	013.06	0.00
7	1	03.5	Ud	28.06	34.97	36.81	Ud	Ud	1.00
	2	25.5	29.87	26.83	24.79	24.58	Ud	Ud	4.12
	3	28.5	28.86	25.38	22.16	22.40	Ud	Ud	3.02
8	1	01.5	28.73	24.87	20.89	22.61	01.00	001.00	1.00
	2	04.5	31.08	27.40	21.74	29.61	01.13	000.35	0.01
	3	25.5	31.84	28.68	22.04	33.86	01.62	000.26	0.00
	4	28.5	32.28	29.77	27.83	29.73	02.54	010.47	0.88
9	1	01.0	31.23	28.68	25.40	25.89	01.00	001.00	1.00
	2	25.0	31.23	28.95	23.07	24.91	01.22	000.20	0.39
	3	88.5	31.57	29.21	23.61	25.83	01.14	000.23	0.30
	4	92.5	31.61	29.74	24.75	26.18	01.61	000.49	0.52
Average		33.33	27.82	22.91	27.34
Standard deviation		02.21	01.17	02.38	02.81

^* FASN, Fatty acid synthase;

^� ACTB, Actin, beta;

^� Ud, Undetermined

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