DOI: 10.6084/m9.figshare.30103729
title: <b>Quantitative PCR Evaluation of Chitinase and Chitosanase Antifungal Effects on Scalp Mycobiome</b>

description: A clinical study (n=18) evaluated the impact of a solution containing 0.25% chitinase (100 U/g) and 0.25% chitosanase (200 U/g) on scalp <i>M.</i> <i>furfur </i>and <i>M. restricta</i> DNA and RNA levels using quantitative PCR (qPCR).A 4×4 cm area was selected on the hairy part of the scalp. Pre-treatment samples were collected from these areas using sterile swabs and placed in microtubes with lysis buffer. Subsequently, 1 mL of the enzyme solution was added to this area. . After a 3-hour incubation, post-treatment samples were collected using sterile swabs and transferred to microtubes with lysis buffer for further analysis.Nucleic acids were extracted using the LIRA reagent (BioLabMix, Russia). Swabs collected from the scalp surface were placed in 1 mL of LIRA reagent and incubated for 10 minutes at room temperature. Chloroform (200 µL) (Sigma-Aldrich, USA) was added to the lysate, followed by vigorous shaking for 5 seconds and incubation for 5 minutes with intermittent manual mixing. The swab was removed, and the mixture was centrifuged at 10.000 × g for 10 minutes at 4 °C. The resulting phases—organic (lower), interphase, and aqueous (upper)—were separated. The aqueous phase was transferred to a new tube for RNA extraction, while the organic phase and interphase were retained for subsequent DNA isolation.For RNA extraction, an equal volume of isopropanol (Sigma-Aldrich, USA) was added to the aqueous phase, mixed by inversion, and incubated for 10 minutes at room temperature. The sample was centrifuged at 12.000 × g for 10 minutes at 4 °C. The supernatant was discarded, and the pellet was washed with 1 mL of 80% ethanol (Sigma-Aldrich, USA). After centrifugation at 12.000 × g for 5 minutes at 4 °C, the supernatant was removed, and the pellet was air-dried for 10 minutes. RNA was resuspended in 15 µL of RNase-free water and stored at -20 °C.For DNA extraction, the organic phase and interphase were combined, homogenized, and mixed with 300 µL of 96% ethanol. After inversion and incubation for 5 minutes at room temperature, the sample was centrifuged at 2.000 × g for 5 minutes at 4 °C. The supernatant was discarded, and the pellet was resuspended in 1 mL of 0.1 M sodium citrate in 10% ethanol (pH 8.5). Following incubation for 30 minutes at room temperature and centrifugation, the pellet was washed with 1 mL of 80% ethanol, incubated for 20 minutes, and centrifuged again. The dried pellet was dissolved in 15 µL of 40 mM NaOH, neutralized with 0.5 µL of 1 M HEPES, and stored at -20 °C.Quantitative PCR (qPCR) was performed using a BioRad CFX96 thermocycler (BioRad, USA) with the Biomaster HS-qPCR mix (BioLabMix, Russia) under manufacturer-recommended conditions. The 50 µL reaction mixture contained 1× RT-qPCR buffer, 0.1 µM of each primer (Table S3), 0.1 µM dNTPs, 2.5% DMSO, 5% RT-qPCR mix, and DEPC-treated water. The ITS region of 26S rRNA was used as reference gene. The primers were designed based on 26S rRNA genes of <i>Malassezia</i> species.The PCR protocol included an initial denaturation at 95 °C for 7 minutes, followed by 40 cycles of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 40 seconds, and elongation at 72 °C for 35 seconds. Primer annealing temperatures were calculated using the Tm Calculator. Real-time monitoring was performed after each elongation step.qRT-PCR was performed using the BioRad CFX96 thermocycler (BioRad, USA) with the qOT-PCR SYBR Blue mix (BioLabMix, Russia), a one-step reverse transcription and real-time quantitative PCR mix containing the SYBR Green I fluorescent dye, following the manufacturer’s protocol. The 50 µL reaction mixture consisted of 1× RT-qPCR SYBR Blue buffer, 0.1 µM of each primer (Table S3), 0.1 µM dNTPs, 2.5% dimethyl sulfoxide (DMSO), 5% RT-qPCR mix, and DEPC-treated water. The primers were designed based on 26S rRNA genes of <i>Malassezia</i> species.The qRT-PCR protocol included a reverse transcription step at 45 °C for 30 minutes, followed by initial denaturation at 95 °C for 7 minutes and 40 cycles of denaturation at 95 °C for 30 seconds, primer annealing at 55 °C for 40 seconds, and elongation at 72 °C for 35 seconds. Primer annealing temperatures were calculated using the Tm Calculator (https://tmcalculator.neb.com). Real-time fluorescence monitoring was conducted after each elongation step.Following PCR amplification, cycle threshold (Ct) values were determined both before and after treatment for each sample, using specific probes for the reference gene and an intercalating dye for the target gene. The analysis was conducted in three sequential steps:Calculation of Transcriptional Changes: The changes in transcript levels for both the reference and target genes were calculated separately:ΔCt (reference gene) = Ct (pre-treatment) – Ct (post-treatment)ΔCt (target gene) = Ct (pre-treatment) – Ct (post-treatment)Normalization of Target Gene Expression: The expression of the target gene was normalized relative to the reference gene using the formula:ΔΔCt = ΔCt (reference gene) – ΔCt (target gene).Quantification of Relative Nucleic Acid Content: The relative change in nucleic acid content was determined using the formula 2^(-ΔΔCt) where ΔΔCt = ΔCt (target sample) – ΔCt (reference sample). For ease of interpretation, the fold change in expression was calculated as 〖1/2〗^ΔΔCt Values greater than 1 indicate a reduction in relative nucleic acid content post-treatment, while values less than 1 indicate an increase.<br>