2.1 Subjects:
For this study in total eighty six (86), clinically confirmed psoriasis vulgaris patients were recruited. This case-control study encompassed histologically confirmed cases of psoriasis vulgaris and an equivalent number of controls. All subjects with psoriasis vulgaris were enrolled between May 2018-February 2020. All cases underwent clinical and histopathological confirmation by a minimum of two dermatologists. Controls consisted of unaffected individuals without a family history of psoriasis. Comprehensive primary clinical data were thoroughly recorded. This included demographic information, age and its emergence (early (≤ 40 years) or late onset (> 40), familial history, severity range of psoriatic skin lesions assessed by PASI (Psoriasis Area and Severity Index) score.
The Vardhman Mahavir Medical College (VMMC) and Safdarjung Hospital, (IEC/VMMC/SIH/Project/2018/1094) Institutional Ethical Committee, granted the approval for this study. Skin biopsies were collected from individuals with psoriasis, adhering to the approved protocol, and every participant in the study provided informed consent. The whole sample collections and experiments were performed adhering to the guidelines and regulations.
2.2 Collection and Extraction of Tissue and RNA:
Out of 86 enrolled patients, peri-lesional skin tissues from 42 cases underwent qRT-PCR (quantitative reverse transcription polymerase chain reaction) for processing of gene expression and immunohistochemistry (IHC). The remaining patients were not included in the study because their RNA quality was low and they were not histopathologically confirmed. 5 mm punch biopsy was obtained from each participant and instantly divided into two fragments. First section was preserved in RNA later and kept for gene expression research at -80°C, while the second section was fixed in buffered formalin for histopathological confirmation. The sample sets were subsequently forwarded to the National Institute of Pathology (ICMR) for additional processing, including histopathological confirmation, mRNA expression analysis, and IHC.
2.3 RNA isolation from tissue samples:
Perilesional skin tissue samples were used to extract RNA using AllPrep DNA/RNA Mini Kit of Qiagen. β- mercaptoethanol (10 µl) was freshly added to buffer RLT Plus (1 ml) just before use. 5–10 mg of biopsy samples soaked in RNA later, and then crushed using mortar and pestle. The resulted tissue’s paste were subsequently moved to 1.5 ml micro centrifuge tubes (MCTs) and mixed with RLT buffer added with β-mercaptoethanol (600 µl) and thoroughly blended. Subsequently, the lysate is transferred to QIAshredder column positioned atop a Qiagen’s collection tube (2ml). Then the samples were centrifuged (13,000 rpm for 2 minutes at RT).
Without interfering with the flow-through, the pulverized lysate was extracted and moved to an AllPrep DNA spin column that was set up on a 2 ml collection tube. One minute of 10,000 rpm centrifugation was carried out at RT. While the flow-through underwent RNA isolation processes, the DNA spin column was utilized for DNA isolation. It was combined with 600µl of 70% ethanol, moved to a RNeasy spin column, and centrifuged for one minute at 10,000 rpm. After that, 700µl of buffer RW1 (reconstitute with ethanol) were used to wash the spin columns, and they were centrifuged. Following two rounds of washing with 500 µl of buffer RPE (reconstituted with ethanol), the columns were centrifuged for two minutes at room temperature at 10,000 rpm to get rid of the ethanol. RNA was extracted from the columns using 50µl of RNase-free water, and the mixture was centrifuged for one minute at 10,000 rpm after the columns were moved to 1.5 ml MCTs.
To boost RNA concentration, the elution procedure was performed again using the same elute. Isolated RNA stored at -80°C and processed further. Thermo Scientific, (Waltham, MA, USA), used the Nanodrop 2000 Spectrophotometer to quantify the eluted RNA.
2.4 cDNA synthesis:
Thermo Scientific's RevertAid First Strand cDNA Synthesis Kit was used to create cDNA from about 1 µg of total RNA. Nuclease-free H2O was used to dilute 1 µg of RNA to a volume of 12 µl. This was then combined with the kit's included oligo dT primers (1 µl), 5X buffer (4 µl), dNTPs (2 µl), RNase inhibitor (1 µl), and reverse transcriptase (1 µl) to create an aggregate volume of 20 µl. The conditions for synthesizing cDNA were as follows: heating the enzymes for five minutes at 65°C, then for sixty minutes at 42°C, denaturing them for five minutes at 70°C, and finally for an infinite hold at 4°C. The final concentration of cDNA was around 50 ng/µl.
2.5 Gene Expression Study by quantitative PCR using Taqman probes:
After diluting the cDNA product five times with ddH2O, approximately 10 ng was utilized for every qRT-PCR assay. Transcripts were quantified using an Applied Biosystems 7900HT Fast Real-Time PCR equipment with a Taqman probe-primer set purchased from Applied Biosystems CARD14 (Hs00364499_ml). In all other gene expression studies, transcripts were quantified using Applied Biosystems' TaqMan Universal PCR Master Mix in a 20 µl reaction with 1 µl of probe. Following that, standard PCR settings were used, which included 10 minutes at 95°C, 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C, and an analysis of the dissociation curve using 15 seconds at 95°C, 15 seconds at 60°C, and 15 seconds at 95°C. A 96-well plate from ABI in Foster City, California was used to run each sample in triplicate. Melting curve analysis using StepOne software v.2.3 (ABI, Foster City, CA) was used to confirm the probe specificity. Gene expression’s normalization was determined by expression fold change of GAPDH gene in the involved skin compared to uninvolved skin of same patient. Using DataAssist software v.3.01 (ABI, Foster City, CA), the mean Ct for each gene was normalized with endogenous control (GAPDH) to get ΔCt. The fold change (FC) was then computed using the 2ΔΔCt approach.
2.6 Immunohistochemistry analysis:
Validation of samples were carried out through IHC, using Novo link Polymer detection systems (Leica Bio systems). Tissue blocks embedded in paraffin and treated in formalin were used to create thin sections with a thickness of 4 µm. Paraffin was removed by dipping the slides inside Coplin jars containing Xylene (Merck) for 30 min (two changes for 15 min). The portions were then gradually rehydrated by being kept for 15 minutes each in Coplin jars with gradients of ethanol (Xylene 95% Ethanol, 70% Ethanol, 50% Ethanol, and lastly dH2O). To extract the antigen, slides were then boiled in a 10 mM sodium citrate solution containing 0.05% Tween 20 for 20 minutes. After that, slides were progressively cooled under running water, cleaned with TBS, and blocked for two hours at room temperature using the Protein Block buffer that included with the kit.
Tissue sections were marked with mini-PAP pen (Invitrogen) to allow for minimum reagent usage. The blocking buffer was decanted, the primary antibody [CARD14 (1:150)] was added and left intact overnight at 4°C in a wet chamber. Slides were rinsed three times with TBS for five minutes each the next day. Peroxidase block was added for 15 min to neutralize endogenous peroxidase activity, followed by wash in TBS 2 times and incubation with post- primary block for 30 min. After wash, polymer was added and incubated for 30 min to allow detection of mouse and rabbit primary antibodies by the polymer system. After washing the polymer by 2 washes in TBS, AEC working solution was added and incubated for 5 min. AEC slides washed in water and were counter-stained for 2 min with hematoxylin. Hematoxylin was washed with water for 5 min, and then slides were gradually dehydrated using grades of ethanol. After incubation in xylene for 15–30 min, slides were mounted using CC mount, allowed to dry and then observed under microscope and image was captured. All the 42 disease sections were histopathologically confirmed as plaque psoriasis/psoriasis vulgaris.