School of Pharmacy, Queen's University Belfast, Belfast, Northern Ireland, UK.
Mol Biotechnol. 2013 Jun;54(2):187-97. doi: 10.1007/s12033-012-9551-6.
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.
两栖动物皮肤分泌物是生物活性肽的独特来源，其供体物种目前正迅速从生物圈中消失。在这里，我们报告说，在将皮肤颗粒腺的肽和多聚腺苷酸化 mRNA 储存在 -20°C 的 0.1%三氟乙酸水溶液中多年后，其刺激皮肤分泌物的储存仍然可以进行研究。经过 12 年储存的冷冻酸化蟾蜍（Bombina variegata）皮肤分泌物溶液解冻后，直接用于反相 HPLC 分级分离。取出额外的样品，快速冷冻并冻干，然后使用磁性寡聚 dT 珠和逆转录进行多聚腺苷酸化 mRNA 捕获，以构建 cDNA 文库。使用在蟾蜍皮肤中发现的蛙皮素和缓激肽肽作为模型，在反相 HPLC 馏分中定位每种类型的单个变体肽，并使用 RACE PCR 策略从 cDNA 文库中克隆其相应的生物合成前体编码 mRNA 转录本。这项研究明确表明，两栖动物皮肤分泌物中的肽及其生物合成前体编码的多聚腺苷酸化 mRNA 在冷冻酸化的皮肤分泌物样本中可以稳定保存相当长的时间，这一发现可能对存档材料的研究具有根本意义，但也对更广泛的分子生物学领域具有意义。
