Ethical clearance and sample size calculation
The subjects were recruited from the Department of Periodontics and Oral Implantology at SRM Dental College, Chennai, outpatient clinic. Patients were enrolled from January 2020 to June 2021 after the Institutional Scientific and Ethical Review Board gave its approval for the research (IEC approval: SRMDC / IRB / 2019 / MDS / No. 506). Specific inclusion and exclusion criteria were used to determine recruitment, and gingival samples were collected for analysis. All participants provided written informed consent following an explanation of the study's objectives, protocol, risks, and potential benefits to them.
Study population, eligibility criteria, and clinical parameters recorded
Sample size was calculated using G Power software based on the article by Radović et al. . The mean values with effect size of 0.8 were used for analysis. To achieve 90% power with 1% α error, a sample size of 98 was required. The statistical significance was set to P≤ 0.01 (α=1%). The study subjects were assigned into 2 groups: group 1 (test): N = 49, systemically healthy patients with localized stage III/IV periodontitis, and group 2 (control): N = 49, systemically and periodontally healthy subjects with no clinical attachment loss.
Group I: Age ≥ 18years, patients with untreated stage III or stage IV periodontitis (according to 2017 EPF/AAP Classification), Gingival Index ≥ 1, periodontal probing depth ≥ 6mm, clinical attachment loss ≥ 3mm
Group II: Age ≥ 18years, systemically healthy, probing depth ≤ 3mm, no clinical attachment loss, Gingival Index score <1, presence of at least 20 natural teeth excluding 3rd molars
Patients with any systemic or autoimmune diseases; patients under medication with immunosuppressants, antibiotics, and anti-inflammatory drugs in the last 6 months; pregnant or lactating mothers; smokers; and patients with previous history of periodontal therapy.
The subjects underwent routine periodontal examination and the clinical parameters were recorded by a single calibrated examiner to avoid bias. The site-specific clinical parameters like Probing Pocket Depth (in mm), Clinical Attachment Level (in mm), Plaque Index (PI) [Silness and Loe, 1964], and Gingival Index (GI) [Loe and Silness,1963] were recorded.
Gingival tissue sample collection
During the study period, there was a complete lockdown declared from March 2020 till Aug 2020, second lockdown was from mid-April 21 till mid-June 21, the institution started functioning post-lockdown with appropriate COVID protection protocols, and the patients were treated for emergency procedures like extractions after they were declared negative in RT-PCR analysis. Further, orthodontic treatment also resumed following appropriate COVID protection protocol and negative RT-PCR report. Gingival tissue samples were collected for group I from hopeless prognosis tooth diagnosed with stage III/IV periodontitis indicated for extraction and for group II from patients scheduled for extraction of periodontally healthy premolars for orthodontic purposes and from those undergoing crown lengthening procedure in a non-inflamed periodontium. Local infiltration was given using 2% lignocaine with 1:80,000 adrenaline and then using 15c blade the gingival tissues were excised using intracrevicular and inverse bevel incisions on the facial/lingual/interproximal gingiva to obtain a single tissue biopsy measuring 3mm × 5mm. The tissues were placed in a 2-ml Eppendorf tube containing RNA later solution and stored at −20°C.
Quantitative real-time PCR analysis
miRNA isolation was performed with the help of a miRNA isolation kit (Invitrogen mirVanaTM, Thermo Fisher Scientific, USA) and phenol. The miRNA DNA synthesis kit (TaqMan™ MicroRNA Reverse Transcription Kit Catalog number: 4366597) was used for preparing the cDNA template to evaluate miRNA expression levels using real time-PCR (Rotor Gene Q, Qiagen, Venlo, Netherlands). miRNA expression master mix (MM) was prepared using TaqMan Advanced miRNA assay according to the manufacturer’s instructions.
The primers used for qRT-PCR are as follows: miRNA.-155: 5′CUGUUAAUGCUAAUGUGUAGGGGUUUUGC3′ (forward), 3′GACAAUUACGAUUAUACAUCCUCAGAACU 5′ (reverse); miRNA-361-5p: 5′ UUAUCAGAAUCUCCAGGGGUAC 3′ (forward) (endogenous control) (Taqman Micro Assay, Thermo Fisher Scientific, USA catalog number:4427975). The RNA isolation procedure for the MITF gene was carried using the RNAiso plus Reagent (TRIZOL) and then cDNA was prepared using High Capacity Reverse Transcription Kit (TaqMan™ High-Capacity cDNA Reverse Transcription Kit, Catalog no:4368814) following manufacturer’s protocol. Gene expression master mix was prepared using SYBR Green dye (Sigma Aldrich KAPA SYBR® FAST Universal 2X qPCR Master Mix, KK4600) for MITF and GAPDH gene expression (endogenous control). The primer pairs used for MITF were 5′-ACTTTCCCTTATCCCATCCACC-3′ (forward), 5′-TGAGATCCAGAGTTGTCGTACA-3′ (reverse); for GAPDH, 5′-TGCACCACCAACTGCTTA-3′ (forward), 5′-GATGCAGGGATGATGTTC-3′ (reverse) primer sets were used. The RT-PCR was carried out for all the cDNA-converted samples in duplicates.
The relative expression of miRNA-155 and MITF was evaluated using Livak method and reported as fold change normalized to the endogenous control gene as follows:
❖ ΔCT = CT of the target DNA − CT of the housekeeping gene.
❖ ΔΔCT = ΔCT value of the sample for a particular target − average ΔCT value of the control group for the same target.
❖ The relative expression (Fold Change) of miRNA = 2−ΔΔCT.