Using Skin Gene Markers for Estimating Early Postmortem Interval at Different Temperatures Mona Mohamed Ali, PhD,* Samah Fathy Ibrahim, PhD,*† and Amel Ahmed Fayed, PhD† Abstract: Many researches document long-term RNA persistence in a variety of tissues and its applicability in estimating the postmortem interval (PMI). Skin-specific mRNA marker, late cornified envelope 1C (LCE1C), was used to identified skin samples. Before using the LCE1C in criminal casework, its persistence and applicability for estimating PMI in different temperatures were tested. Twelve skin samples were collected from 6 patients, and 6 samples were kept at 24°C and others were kept at 40°C for 5 days. The expression levels of LCE1C mRNA are serially detected and quantified using real-time polymerase chain reaction. The expression levels of LCE1C were decreased with increasing the time interval in time-dependent manner, whereas changing the surrounding temperatures did not show any statistical significance. These results could suggest using LCE1C in estimation of PMI. Moreover, these encourage investigators and crime laboratories to know environmental conditions before interpreting the results. Key Words: forensic sciences, LCE1C mRNA, skin, postmortem interval, RT-PCR (Am J Forensic Med Pathol 2017;00: 00–00) D etermination of the time of death, postmortem interval (PMI), is an important consideration in forensic practice. Physical (algor mortis, livor mortis), physicochemical (rigor mortis), bio- chemical (electrolyte concentration, enzyme activity), microbio- logical (decomposition), entomological, and botanical processes were used to estimate PMI. 1 Applying these methods to estimate early PMI is of limited practical relevance. 2 In addition, many in- trinsic factors (age, sex, physiological, pathological conditions, etc) and extrinsic factors (temperature, humidity, insect activity, etc) affect them. Thus, there is a trend in forensic science to de- velop an alternative approaches to determine PMI using DNA and RNA molecular technologies. 3 The developments in forensic mRNA profiling systems use the RNA rapid decay rate in determination of PMI if the residual quantity of RNA correlates with the elapsed time. 3 Hanson et al 4 identified late cornified envelope (LCE) mRNA especially LCE1C mRNA as a highly sensitive and abun- dant skin biomarkers. Abd El Razik et al 5 showed that the highest LCE1C expression was found after 1 day interval, whereas the lowest value was found after 3 weeks interval. Moreover, they stated that LCE1C disappeared at fourth week. Previous studies tested the relationship between RNA and PMI through detection of poly A percentage in total RNA, 6 quotient areas in certain genes, 7 or cycle threshold value of a certain gene. 8 To the best of our knowledge, the effect of exter- nal factors, for example, temperature on decay rate of mRNA markers, which can be used for PMI determination, was not fully tested. Our hypothesis is to test the ability of using LCE1C mRNA expressional decay in estimating early PMI and evaluate the effect of temperature on it. MATERIALS AND METHODS Twelve healthy surgical skin specimens from 6 volunteers were used after obtaining an informed written consent from each participant. Any subject that has any pathological skin lesion was excluded from the study. A skin specimen measuring 3 Â 2 Â 1.5 cm with an average weight of 50 g was obtained from lower abdominal area and kept in sterile container. The received time was recorded as day 0. The half of skin specimens (group 1) was kept at 25°C (room temperature), and the other half (group 2) was kept at 40°C (kept in oven). Moreover, both groups were exposed to dried air. The expression decay level of LCE1C mRNA was detected at 0, 1, 2, 3, 4, and 5 days as Abd El Razik et al 5 concluded that the highest LCE1C mRNA expression values were detected at first week. After each time interval had passed, approximately 100 mg was cut from each skin sample, immedi- ately frozen in lysis buffer, and stored at -80°C until experimental analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as endogenous reference housekeeping gene to elim- inate any changes in mRNA levels caused by sample processing that might alter the target gene expression activity. 9 The research protocol was approved by the ethical committee of the Faculty of Medicine, Cairo University. The expression levels of LCE1C mRNA required the following: Total RNA Extraction The isolation of total RNA was done using the total RNA ex- traction kit (VIVANTIS nucleic acid extraction kit, Malaysia), and then its quantity (ng/μL) was measured using an ultraviolet/visible spectrophotometer (UItrospec 4300 pro, Biochrom, Cambridge, UK). BioRT 1-step real-time polymerase chain reaction (RT-PCR) kit cat. no. BSB07S1 was used to perform reverse transcription ac- cording to manufacturer's instruction. Real-Time Fluorescent Quantitative PCR Primers were designed based on sequences obtained from GenBank, which were imported into Allele ID 6 software (Pre- mier Biosoft International). The assay setting “SYBR Green De- sign” was chosen to limit primer sequences to regions of the little secondary template structure. Then, the designed primer se- quences were validated on BLAST to have a high efficiency. Primers were obtained from Takara Biotechnology. The sequences, length of production, and source sequences according to Bauer et al 10 were as follows: Manuscript received February 27, 2017; accepted June 12, 2017. From the *Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Cairo University, Egypt; and †College of Medicine, Princess Nourah Bint-Abdulrahman University, Saudi Arabia. The authors report no conflict of interest. Reprints: Samah Fathy Ibrahim, PhD, Forensic Medicine and Clinical Toxicology Department, Faculty of Medicine, Cairo University, Cairo 11562, Egypt. E-mail: samahibraheem@yahoo.com. Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0195-7910/17/0000–0000 DOI: 10.1097/PAF.0000000000000337 ORIGINAL ARTICLE Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017 www.amjforensicmedicine.com 1 Copyright © 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.