This prospective cohort study was conducted at The Biochemistry Department Laboratory, Faculty of Medicine, Cairo University. The participants included in our study were healthy volunteers of both sexes aged 18–40, and their saliva, nasal, and menstrual blood swabs were obtained. All individuals with blood diseases, malignancies, and respiratory tract diseases were excluded from our study.
Consent
The participant consent was verbal and was approved by the Local Ethical Committee of Forensic Medicine and Clinical Toxicology Department (ethics reference: I-070318). The swabs were considered a non-invasive procedure, and consent can be expressed verbally.
Design of work
The present study included 180 samples from 40 volunteers: 20 females (26–30 years old) for menstrual blood collection and 10 males plus 10 females (26–30 years old) for nasal and salivary sample collection. For each fluid, 60 samples were collected (20 samples were dried at ambient temperature for 1 day before being stored at −20°C), 20 samples were dried at room temperature for 3 days before being stored at −20°C, and 20 samples were dried at room temperature for 7 days before being stored at −20°C to be studied later. For each body fluid, we intended to detect two specific markers (for the saliva, STATCH and HTN3; for nasal secretions, STATCH and BPIFA1; and for the menstrual blood, MMP7 and MMP10) and a housekeeping gene (B-actin) as an internal control.
Extraction of RNA from the saliva, nasal, and menstrual blood swabs
The extraction of intact RNA needs four steps: disruption of cells, denaturation of nucleoprotein complexes, endogenous ribonuclease (RNase) activity inactivation, and removal of DNA and proteins that contaminate the samples. The purification of RNA contaminants was performed using simple washing steps with 70% alcohol. They were then eluted by centrifuge at full speed (14,000 rpm or 10,000× for 2 min) and then stored at −20°C. Isolation reagent supported by Ambion, Inc.
Reverse transcription and PCR
The yield of the total RNA of the collected samples was determined at 260 and 280 nm using Beckman dual spectrophotometer. Gene expression was measured using One-Step Taq qRT-PCR Green Master (Ambion, Inc) in a real-time PCR instrument (StepOne, version 2.1, Applied biosystem, Foster City, USA). For qRT-PCR, we developed a triplex which would include 2 body fluid-specific markers and a housekeeping gene (B actin) as an internal control. Ten μL of the total RNA from each sample was used for RT-reaction and amplified in a total reaction volume of 25μL. The standard reaction mixture contained buffer (10 mM Tris-HCl, pH 8.3, 50mM KCl, and 1.5mM MgCl2), 0.5 mM dNTP mix, 0.4 μM of each PCR primer Table 1 and 1.25U AmpliTaq Gold® DNA polymerase. The thermal profile was as follows: 45°C for 15 min one cycle (for cDNA synthesis), 10 min at 95°C for reverse transcriptase enzyme inactivation followed by 50 cycles PCR amplification were programmed. Each cycle was 10 s at 95°C for CDNA denaturation, 30 s at 60°C for primers annealing, and 30 s at 72° for Taq polymerase extension. Normalization of each target gene was calculated relative to values of β-actin housekeeping gene, and the result was analyzed with the Step One Applied Biosystem software.
Statistical analysis
Data were coded and entered using the Statistical Package for the Social Sciences (SPSS) version 26 (IBM Corp., Armonk, NY, USA). Data were summarized using mean and standard deviation. The comparison between the different genes in the same sample was made using a paired t test. The comparisons between the different samples were made using an unpaired t test when comparing two samples, and the analysis of variance (ANOVA) with multiple-comparisons post hoc test was used when comparing more than two samples (Chan 2003). A p value of less than 0.05 was considered statistically significant.