Reverse transcriptase enzyme

RNA was isolated using the TRIzol RNA reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. The RNase-Free DNase Set (Qiagen) was used to eliminate any contamination of the genomic DNA. For the synthesis of complementary DNA , total RNA (1 µg) was reverse transcribed using random primers and the reverse transcriptase enzyme (Applied Biosystems, Foster City, CA). Subsequently, qPCR was performed using the Power SYBR Green PCR Mastermix (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 7500 Fast Real-time PCR System. The reaction was conducted in triplicate for each gene. The human gene-specific primers are listed in Table 1.

Whole head samples were coronally cryosectioned at 60 μm until the region of interest (ROI) was visible and microdissected. Tissue samples collected for mRNA analysis included the mouth, eye, cortex, hippocampus, hypothalamus, and trigeminal ganglia. ROIs were removed via microdissection with a scalpel and homogenized in Trizol (Invitrogen 15596-026, CA, USA). RNA was isolated with chloroform extraction and alcohol precipitation. RNA quantity was measured using a Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA) and RNA integrity was evaluated by agarose electrophoresis. cDNA was synthesized from DNase-treated RNA with reverse transcriptase enzyme (Applied Biosystems 4319983, CA, USA) and primer (Applied Biosystems 4319979, CA, USA). SYBR (Bio Rad 170-8882, CA, USA) was used along with the primers listed in Supplemental Table 2 to detect Avpr1a. Primers targeted the Avpr1a transcript NM_016847.2 exon 1 at 1,241–1,330 bp and exon 2 at 1548–1619 bp. AVPR1A KO mice only have genomic exon 2, as exon 1 is removed^{18 (link)}.

Total RNA was extracted from lung tissues using TRIzol reagent (Invitrogen; Carlsbad, CA) and reverse-transcribed into cDNA using reverse transcriptase enzyme (Applied Biosystems; Foster City, CA). The PCR reaction was performed in 20 μl of final volume containing 0.08 μM of forward and reverse primer, 2 μl of 10–20× diluted original cDNA, and 10 μl SYBR Green PCR Master Mix (Applied Biosystems) using Applied Biosystems 7300 real-time PCR machine. Mouse β-actin served as an internal control gene for normalization. Relative expression of mRNA was represented as fold change in comparison to the sham group. The sense and anti-sense primer sequences of mouse genes are, IL-6 (NM_031168): 5’-CCGGAGAGGAGACTTCACAG-3′ and 5′-GGAAATTGGGGTAGGAAGGA-3′; IL-1β (NM_008361): 5’-CAGGATGAGGACATGAGCACC-3′ and 5’-CTCTGCAGACTCAAACTCCAC-3′; tumor necrosis factor-α (TNF-α) (NM_013693.2): 5′-AGACCCTCACACTCAGATCATCTTC-3′ and 5′-TTGCTACGACGTGGGCTACA-3′; interferon γ (IFNγ) (NM_008337): 5’-GGCTTTGCAGCTCTTCCTC-3′ and 5’-CCAGTTCCTCCAGATATCCAA-3′; IL-10 (NM_010548): 5’-CAGCCGGGAAGACAATAA CT-3′ and 5’-GCATTAAGGAGTCGGTTAGCA-3′; MIP-2 (NM_009140): 5’-CCCTGGTTC AGAAAATCATCCA-3′ and 5’-GCTCCTCCTTTCCAGGTCAGT-3′; β-actin (NM_007393): 5’-CGTGAAAAGATGACCCAGATCA-3′ and 5’-TGGTACGACCAGAGGCATACAG-3′.

For the isolation of RNA, GSCs (3.2 × 10⁵/well) were seeded in 6‐well culture plate and 24 hours later treated with 6.8 μg/mL of K rotunda tuberous rhizome‐mediated Ag/AgCl‐NPs as described above. Cells incubated without Ag/AgCl‐NPs were used as control. For the isolation of RNA, cells were washed by PBS and dissolved in Tri‐reagent (Sigma, USA). Then by using trichloromethane, isopropanol and 70% ethanol in different steps RNA was isolated and collected in DNases and RNases free water. After purification of RNA, 18s gene was used for checking the quality and as references to normalize the qPCR data. cDNA was synthesized from an equal amount of Ag/AgCl‐NPs treated and untreated RNA by reverse transcriptase enzyme as described by the manufacturer (Thermo scientific). Samples were prepared by the addition of cDNA, forward and reverse primers (Supplementary Table‐1), water and 2 × Syber master mix as described by the manufacturer (Applied Biosystems). Bio Rad thermal cycler was used for qPCR and the condition was set to 50 and 95°C for 2 minutes each and for 40 cycles, 95°C for 15 seconds and 60°C for 1 minute. Finally, real‐time PCR data were analysed by double delta CT methods using Excel software where 18s was used as reference gene to normalize the data.