The first round of PCR involved 10×UPM, outer gene-specific primers and PCR Polymerase (TransGen).
Then, two PCR amplifications are preformed per library: the first uses the outer adaptor primer (AP1, provided by the manufacturer) provided in the kit and the outer, gene-specific primer (GWRUT C30gsp1; Table 1).
An outer gene-specific reverse primer located in exon 5 and a nested primer located in exon 4 were used for amplification, obtaining a fragment of about 0.5 kb as product of the nested PCR.
PCR was performed with the inner primer towards the ligated adaptor sequence (provided in RLM-RACE kit, Ambion) and the outer gene-specific primer (Table 1) designed downstream of the predicted stop codon for svPINK1.
The first found PCR was carried out with 5'RACE general PCR and outer gene-specific PCR, and the next PCR was run using the diluted initial PCR reaction and inner 5' RACE and gene-specific primers (GSP).
All the genes in a functional module were designated inner genes, and those outside a functional module were designated outer genes.
Then 1 μl of reverse transcription product was used as template to amplify the 5' end of the potential target by using the outer 5' RNA adaptor primer supplied in the kit and outer 3' gene-specific primer.
L in was the number of links between inner genes, G in was the number of inner genes with links to other inner genes, L out was the number of links between G in inner genes and outer genes.
G out was the number of outer genes with links to G in inner genes, If C in was greater than C out, we recognized the functional module as compact.
These genes include the antigenic outer membrane gene porB, pilin glycosylation genes pglC and pglD, iron transporter substrate-binding protein gene fetB, transferrin binding protein gene tbpB, and DNA-binding/iron metalloprotein/AP endonuclease gene gcp.
The INNER NO OUTER (INO) gene, which encodes a YABBY-type transcription factor, specifies and promotes the growth of the outer integument of the ovule in Arabidopsis.
