Frozen seeds were Belinostat chemical structure pulverized under liquid nitrogen. Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, California, USA) with modifications of the company’s instructions due the high polysaccharide content of the grains and treated with DNase I (RNase-free) (Ambion, Austin, Texas, USA) to eliminate any DNA contamination. For the gene expression analysis, RNA was purified using the RNeasy mini kit (Qiagen,Valencia, California, USA). First-strand cDNA was synthesized from 2 ��g of total RNA, using oligo (dT) primer, random nanomers and M-MLV reverse transcriptase (Invitrogen, Carlsbad, California, USA) in 20 ��L total volume according to the manufacturer’s instructions. cDNA samples were diluted with an additional 80 ��L of milliQ water.

Amplification of gliadin-like avenin genes Three primers, one forward and two reverse (AvenG-1F, AvenG-1R, AvenG-2R), were developed for the amplification of oat prolamin genes (Table 1). These primers were designed based on avenin-coding sequences/regions (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”DQ370180″,”term_id”:”86610883″,”term_text”:”DQ370180″DQ370180, “type”:”entrez-nucleotide”,”attrs”:”text”:”M38722″,”term_id”:”166554″,”term_text”:”M38722″M38722, “type”:”entrez-nucleotide”,”attrs”:”text”:”M83381″,”term_id”:”166556″,”term_text”:”M83381″M83381, “type”:”entrez-nucleotide”,”attrs”:”text”:”J05486″,”term_id”:”166567″,”term_text”:”J05486″J05486, “type”:”entrez-nucleotide”,”attrs”:”text”:”M38721″,”term_id”:”166552″,”term_text”:”M38721″M38721, “type”:”entrez-nucleotide”,”attrs”:”text”:”CK780254″,”term_id”:”42745932″,”term_text”:”CK780254″CK780254, and “type”:”entrez-nucleotide”,”attrs”:”text”:”CK780283″,”term_id”:”42745961″,”term_text”:”CK780283″CK780283).

cDNA PCR amplifications with primer combinations AvenG-1F/AvenG-1R and AvenG-1F/AvenG-2R were conducted in 25 ��L reaction volume consisting of 2 ��L cDNA dilution, 0.2 mM dNTPs (Promega, Madison, Wisconsin, USA), 0.2 ��M of each primer, 2 mM MgCl2, 1X PCR buffer and 2 units of Taq DNA polymerase (Bioline, Boston, Massachusetts, USA). The cycling parameters were 28 cycles of 94��C for 15 s, 68��C for 30 s (decreasing 0.5��C each cycle), and 72��C for 1 min, followed by 30 cycles of 94��C for 15 s, 56��C for 30 s, and 72��C for 1 min. The electrophoretic separation of amplified products was conducted on 1% agarose gel in 0.

5X Tris-borate-EDTA (TBE) buffer. Table 1 PCR primers used for cloning, characterization and quantification of avenin genes. Cloning and sequencing of PCR products and sequence analysis The resulting products of PCR amplification were subjected to ligation into the pGEM-T Easy vector (Promega, Madison, Wisconsin, USA), and introduced Batimastat into competent Escherichia coli (DH5��) cells by heat shock transformation. The positive colonies were amplified using M13 universal primers to identify the clones with an insert.