Single-cell RT-PCR for Spinal Neuron Analysis

Single-cell RT-PCR for spinal neurons was performed as previously described61 (link). Briefly, the contents of dissociated DRG neurons were harvested into patch pipettes with tip diameters of about 20 μm, gently put into reaction tubes containing Dnase I, then were kept at 37 °C for 40 min and followed by 80 °C for 10 min to remove contamination of genomic DNA. After adding reverse transcriptase (SuperScript III Platinum, Invitrogen), the samples were mixed gently and incubated at 50 °C for 50 min. The reaction was stopped by heating sample to 70 °C for 15 min. The cDNA products were used in gene-specific nested PCR. The sequences of the primers are shown in Table 1. The first round PCR was carried out using FastStart Universal SYBR Green Master (Roche, Switzerland). The following PCR conditions were used: 1 cycle of 3 min, 94 °C; 35 cycles of 15 s, 95 °C; 15 s, 60 °C; 1 cycle of 10 min, 72 °C. The second round of PCR was performed using 0.5 μl of the first PCR product as the template. The amplification regents and procedure for the inner primers was the same as that of the first round. A negative control was obtained from pipettes that did not have cell contents but were submerged in the bath solution. The PCR products were displayed on GelRed-stained agarose gels (3%).