Iqtm sybr green supermix

Total bacterial counts of K-O and K-I samples were estimated through quantitative polymerase chain reaction (qPCR). Purified DNA (2 μL) was added to 2× iQTM SYBR^® Green Supermix (BioRad, Mississauga, ON, Canada) along with universal 16S rRNA bacterial primers (300 nM Forward 5′-CCTACGGGNBGCASCAG-3′; Reverse 5′-GACTACNVGGGTATCTAATCC-3′). The total reaction volume was made up to 20 μL with nuclease-free water and the qPCR was accomplished on the CFX96^TM deep well thermal cycler (BioRad, Mississauga, ON, Canada). Standard Escherichia coli DNA (2.4 × 10⁶ cells/mL) and nuclease-free water were used as a positive control (PC) and negative controls (NTC), respectively. Serial dilutions (1:10 × 6) of PC were also amplified along with the samples to obtain a standard curve (R² = 0.650; slope = −3.123; y-int = 38.971). The cycling conditions were set for initial denaturation at 95 °C (3 min); followed by 40 rounds of denaturation at 95 °C (20 s); annealing and extension at 60 °C (45 s). A PCR efficiency of 100.0% was achieved. The melt curve analysis was carried out at 60 °C. The cycle threshold (C_T) values of samples were plotted on the standard curve, to quantify the target gene (16S rRNA gene). This was performed in CFX Maestro^TM software (BioRad). The copy numbers of the target gene were then converted to bacterial cells per m⁻³ of air [18 ].

Splenocytes, ALN cells, and BLN cells (1 × 10⁶) from mice were activated ex vivo by incubation with anti-CD3 (1 μg/mL, BD) for 2 days, and total RNA was isolated from each sample using TRIzol (Invitrogen, Carlsbad, CA, USA). Skin samples were homogenized and centrifuged at 12,000g for 10 min, and the total RNA was extracted from the biopsies of the back skin using TRIzol reagent. RNA was transcribed to cDNA at 42°C for 1 h in a total reaction volume of 25 μL, which contained 5× RT buffer, 10 mM dNTPs (200 units), MMLV-RT (Moloney murine leukemia virus reverse transcriptase), and 100 pmol oligo-dT primer. The cDNA was then used for quantitative real-time PCR with 2× iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) to determine the mRNA levels of IL-17A, IL-22, IL-12p40, IL-23p19, RORγt, human IL-8, human IL-36γ, human CCL20, and GAPDH. To confirm PCR specificity, the PCR products were subjected to melting curve analysis. The comparative threshold method was used to calculate the relative amount of mRNA in the experimental samples compared with the control samples. Gene expression was normalized to the expression of GAPDH.

The quantification of the known and novel miRNAs were carried out using SYBR Green-based real-time PCR. Reverse transcription was performed with RevertAid First Strand cDNA synthesis Kit (Thermo, US) containing 100 ng template RNA, 4 µl of 5×Reaction buffer, 1 µl of Ribolock RNAse inhibitor (20 U/µl), 2 µl of 10 mM dNTP Mix and revertAid M-MuLV reverse transcriptase (200 U/µl) in each reaction (20 µl). The RT reaction was conducted in accordance to the protocol. The PCR reaction contained 0.4 µl of diluted cDNA, 5 µl 2×iQTM SYBR Green supermix (BioRad), 0.2 µl of 10× miScript universal primer and 1 ul of 10× miScript primers. Amplification was performed using CFX96 Real-Time PCR detection system (BioRad) as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. Each RT- PCR reaction was performed in triplicate. Relative expression was calculated using the comparative Ct method while the U6 snRNA was used as the housekeeping gene.

RNA isolation and qRT-PCR were done as described previously [21 (link)]. Real-time PCR (qPCR) and gene specific primers are detailed in Supplementary Table S2. qPCR reactions were done using 25 ng of cDNA/reaction and 2×iQTM SYBR Green Super mix (Bio-Rad, Hercules, CA, USA) and for 25 cycles, unless described otherwise.