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. 2016 Jun 28;6:28791. doi: 10.1038/srep28791

Figure 9. Confirmation of miR395-mediated cleavage of NtSULTR2 mRNA.

Figure 9

RLM-RACE (T4-RNA ligase mediated amplification of 5′ cDNA ends) was conducted to confirm the cleavage of NtSULTR2 mRNA. Total RNA samples were isolated from two weeks old transgenic tobacco. 44 bp RNA adapter was ligated to the purified RNA by using T4 RNA ligase. Adapter-linked RNA was then used to synthesize first strand cDNA, followed by amplification of 5′ ends using the forward primer ASP and the reverse primer GSP. The 589 bp product from the first round PCR was then used as template for the second round PCR using the forward nest primer NASP and the reverse nest primer NGSP, producing a 493 bp second round PCR product. M: DNA molecular weight marker. OE: overexpression line. Red lines indicate miR395 cutting site.