Sybr green 1

Trizol reagent (Tiangen Biotech, China) was employed to extract the total RNA and cDNA synthesis performed using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, United States). qRT-PCR analysis for the expression of NCKAP1 was performed in triplicate using SYBR Green I (Tiangen Biotech, China) in accordance with the prescribed protocol of the manufacturer. The internal control was GAPDH. The primer sequences for NCKAP1 and GAPDH were as follows:

NCKAP1

 5′-TCCTAAATACTGACGCTACAGCA-3′(forward)

 5′-GCCTCCTTGCATTCTCTTATGTC-3′(reverse)

GAPDH

 5′-GTCTCCTCTGACTTCAACAGCG-3′(forward)

 5 ′-ACCACCCTGTTGCTGTAGCCAA-3′(reverse)

Total RNA was extracted using RNA isolation kit (Tiangen Biotech) and subjected to cDNA synthesis using PrimeScript RT reagent Kit (Takara Bio). The cDNA was then used for the evaluation of the relative mRNA levels of the indentified gene, running in an ABI 7300 analyzer (Applied Biosystems). SYBR Green I (Tiangen Biotech) was used as the fluorescent probe. Primer sequences for RT-PCR are listed in the supplementary table 3. The relative expression levels of the target genes were referred to as a housekeeping gene, GAPDH.

After the calcein-AM cytotoxicity assay, total RNA from mixed cells was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Granzyme B and perforin mRNA were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis using SYBR^® Green I with the Real Master Mix kit (Tiangen, Beijing, China). Reactions were run in triplicate and repeated in three independent experiments using the CFX real-time PCR system (Bio-Rad, USA) with cDNA template in a 25 μL reaction under the following conditions: 95°C for 2 min followed by 45 cycles of 95°C for 15 s, 65°C for 15 s and 72°C for 40 s. The primers used for real-time PCR are listed in Table 1 as described by Nagai K et al. [52 (link)].

Total RNA was extracted from kidney tissues and HK-2 cells. The isolated RNA was reverse-transcribed using a miRcute Plus miRNA First-Strand cDNA Kit (Tiangen, China) according to the manufacturer’s instructions. Briefly, the samples were first added to lysate 1 (100 μl) and lysate 2 (600 μl), placed on the purification column, and centrifuged at 12,000 rpm for 30 s, then added the washing solution and centrifuged at 12,000 rpm for 30 s, three times; finally, DEPC water was added and centrifuged at 12,000 rpm for 30 s to obtain the total RNA. The quantity of miRNA was measured using SYBR Green I (Tiangen, China) and a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). The relative expression levels of miR-874-3p were calculated by the comparative cycle threshold method using the expression of U6 small nuclear RNA as a reference for miRNA (sequence-specific primers are shown in Additional file 2: Table S1, S2). The 2^−ΔΔCt method was used for the analysis. The sequence-specific primers used for miR-874-3p and U6 are shown in Additional file 2.

The mRNA levels of ZBTB20, TRPA1, TRPV1, and TRPM8 were analyzed by RT-PCR. The mice were decapitated, and the bilateral TGs were collected with sterilized instruments 30 min after histamine and CQ administration into bilateral cheeks. Total RNA was extracted with an RNA Extraction Kit (Takara). Isolated RNA was reverse-transcribed to synthesize first strand cDNA using a cDNA synthesis kit (Tiangen). The ABI 7500 Real-Time PCR System and SYBR Green I (Tiangen) were used for PCR. Real-time PCR mixtures were prepared, and the reaction conditions were set following the kit instructions. GAPDH was served as an internal control. The melting curve was used to evaluate the reliability of the PCR results. The threshold cycle (CT) value (the inflection point of the amplification curve) was determined, and the relative expression of target genes was calculated using the 2^−ΔΔCt method. The primer sequences for ZBTB20, TRPV1, TRPA1, TRPM8, and GAPDH are shown in Table 1.