Mirvana mirna extraction kit

Total RNA of podocytes was prepared by using mirVana miRNA extraction kit (Ambion). Reverse transcription was performed using the kit from TakaRa (DRR037A). The primers of TLR9 were 5′-CCGTGACAATTACCTGGCCTTC-3′ (forward) and 5′-CAGGGCCTTCAGCTGGTTTC-3′ (reverse); IL12 primers: 5′-GGCCGTCAGCAACATGCTCCA-3′ (forward) and 5′-GGCACAGGGCCATCATAAAAGAGGT-3′ (reverse); 18 s rRNA: 5′TTCTCGATTCCGTGGGTGG-3′ (forward) and 5′-AGCATGCCAGAGTCTCGTTC-3′ (reverse). SYBR Green dye was used in the qPCR. The thermal condition was 95 °C/30 s for denature, followed by 40 cycyles of 95 °C/5 s − 60 °C /30 s on ABI 7900HT Fast Real time System. Threshold cycle (CT) values were determined and the relative abundance of the mRNA was calculated with the formula 2^−△△Ct.

The hydroponic experiment was carried out again using Guiyan 1 and Yunyan 2 under control and 50 μM Cd treatments with 3 replicates. After 5 d Cd treatment, total RNAs were isolated from tobacco roots using TRIzol (Invitrogen, CA, USA) as described above. Small RNA was extracted using the mirVana miRNA Extraction Kit (Ambion, TX, USA). Quantitative real-time PCR (qRT-PCR) was performed using the CXF96 System (Bio-Rad, CA, USA) with a SYBR Green Supermix (Takara, Dalian, China) according to the manufacturer’s instructions. One of the uniformly expressed 5.8S rRNAs was used as the internal control for the stem-loop qRT-PCR. The PCR primer concentration was 10 μM. The reaction conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 10 s, and a melting curve analysis was generated to verify the specificity of the PCR amplification. Each experiment was replicated three times. The relative expression level was calculated using the 2^−ΔΔCT method41 (link), and the fold change = log₂ (2^−ΔΔCT). The miRNAs with fold changes ≥1.5 or ≤−1.5 were considered to be up- or down-regulated in response to Cd stress, respectively. Stem-loop RT primers were designed according to previous work42 (link)43 (link), and were used for the reverse transcription of miRNA (Supplemental Table S7).