title: Parallel peptidome and transcriptome analyses of amphibian skin secretions using archived frozen acid-solvated samples.
abstract: Amphibian skin secretions are unique sources of bioactive peptides and their donor species are currently rapidly disappearing from the biosphere. Here, we report that both peptides and polyadenylated mRNAs from skin granular glands remain amenable to study in samples of stimulated skin secretions following their storage in 0.1 % aqueous trifluoroacetic acid at -20 °C for many years. Frozen acidified solutions of toad (Bombina variegata) skin secretions, stored for 12 years, were thawed and samples removed for direct reverse phase HPLC fractionation. Additional samples were removed, snap frozen and lyophilised for construction of cDNA libraries following polyadenylated mRNA capture using magnetic oligo-dT beads and reverse transcription. Using the bombesin and bradykinin peptides found in bombinid toad skin as models, individual variant peptides of each type were located in reverse phase HPLC fractions and their corresponding biosynthetic precursor-encoding mRNA transcripts were cloned from the cDNA library using a RACE PCR strategy. This study illustrates unequivocally that both amphibian skin secretion peptides and their biosynthetic precursor-encoding polyadenylated mRNAs are stable in frozen acid-solvated skin secretion samples for considerable periods of time-a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.
authors.affiliation: School of Pharmacy, Queen's University Belfast, Belfast, Northern Ireland, UK.
authors.forename: Bing
authors.initials: B
authors.lastname: Bai
authors.forename: Yingqi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Mei
authors.initials: M
authors.lastname: Zhou
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Sijia
authors.initials: S
authors.lastname: Ding
authors.forename: Tianbao
authors.initials: T
authors.lastname: Chen
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Chris
authors.initials: C
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D000663:Amphibians / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001839:Bombesin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001920:Bradykinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D005615:Freezing; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000378:metabolism*
chemical_list: D010455:Peptides; D012322:RNA Precursors; D012333:RNA, Messenger; D001839:Bombesin; D001920:Bradykinin