Primer premier 5

qRT-PCR was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). The reaction mixture contained the Syber Green Master Mix (Bio-Rad) and primer sets. Human β-actin, TRPV1 and α_1D-AR primers designed with Primer Premier 5 (Bio-Rad, Hèrcules, CA) and purchased from SigmaAldrich. Primers sequences were: β-actin: forward 5′-ATCAGCAAGCAGGAGTATGACG -3′; reverse 5′-AAAGCCATGCCAATCTCATCTG-3′; TRPV1: forward 5′ -CTGATGGCAAGGACGACTACC-3′; reverse: 5′ -TTGACCGCAGGGAGAAGCTC-3′; α_1D-AR: forward 5′ -GGTCGTAGCCCTGGTGGTG -3′; reverse: 5′ -CGGAGGAGAAGACAGCGTAGC -3′. Each amplification consisted of heat activation for 15 min at 95°C followed by 40 cycles at 95°C for 10 sec and 60°C for 50 sec. All samples were assayed in triplicate in the same plate and in three different experiments. Measurement of β-actin levels was used to normalize mRNA contents. TRPV1 and α_1D-AR levels were calculated by the 2^-ΔΔCt method and expressed as relative fold respect to control levels.

Overnight bacterial cultures were diluted 1:100 into fresh LB and grown to an OD₆₀₀ of 1 in LB. Three hundred microliters of the bacterial cells was collected by centrifugation at 10,000 × g for 1 min, followed by RNA isolated with a bacterial total RNA kit (Zomanbio, Beijing, China). Qualities of the RNA samples were determined by measuring OD₂₆₀/OD₂₈₀ and OD₂₆₀/OD₂₃₀. Samples with both ratios of ≥2.0 were used for following experiments. A 1.0-μg amount of RNA was used to synthesize cDNA with reverse transcriptase and random primers (TaKaRa, Dalian, China). The same amount of cDNA was mixed with the SYBR II green supermix (Bio-Rad, Beijing, China) and specific primers (Table S2). Real-time PCR was performed with the CFX Connect real-time system (Bio-Rad, USA). Since (p)ppGpp has been shown to inhibit the expression of rRNA and ribosomal protein genes (52 (link)), we used previously reported housekeeping gene PA1805 as the internal control for normalization (53 (link)). Primers were designed with a software program (Primer Premier 5; Bio-Rad, USA), and the sequences ae listed in Table S2. Melting curve analyses were performed with the CFX Connect real-time system (Bio-Rad, USA) to verify the specific amplification of target regions. Each sample was tested in triplicate.