Ecodry kit

Total RNA from radish and tobacco leaves was prepared using TRIzol Reagent (Invitrogen), and first-strand cDNAs were generated using the cDNA EcoDry Kit (Clontech). The qPCR conditions and gene-specific primers, with the exception of those for tobacco NtAN1 and NtAN2, were as described in previously (Lim et al., 2016a (link),b (link)). The qPCR primers for NtAN1 and NtAN2 were as follows: NtAN1 (F, 5′-ACCATTCTCGAACACCGAAG-3′; R, 5′-TGCTAGGGCACAATGTGAAG-3′) and NtAN2 (F, 5′-GTAGACTTCCTGGAAGGACGGCAA-3′; R, 5′-GGCCGAGGTCTGAATATGGTGATC-3′). Gene expression was normalized using the RNA polymerase-II (RPII) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes for radish and tobacco, respectively, as internal references. Three biological replicates were examined for each sample.

For reverse transcription polymerase chain reaction (RT‐PCR) amplification of mRNA and direct sequencing, total RNA was isolated from peripheral leukocytes using RNAlater RNA Stabilization Reagent (Qiagen Inc.) and the RNA was then reverse transcribed into complementary DNA (cDNA) using an EcoDry Kit (Clontech Laboratories, Inc.). RNA from urine sediments were isolated as previously described (Fu et al., 2016; Kaito et al., 2007).

Total RNA was extracted using RNasey mini kit (QIAGEN). Total 1 ug RNA was used to synthesize cDNA using RNA to cDNA EcoDry kit (Clontech). Relative expression determined by standard ∆∆Ct method.