RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine des sciences de la santé, Pavillon de Recherche Appliquée au Cancer, Université de Sherbrooke, 3201 rue Jean--Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine des sciences de la santé, Pavillon de Recherche Appliquée au Cancer, Université de Sherbrooke, 3201 rue Jean--Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
J Virol Methods. 2017 Dec;250:29-33. doi: 10.1016/j.jviromet.2017.09.023. Epub 2017 Sep 22.
5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR).
5' RNA 连接酶介导的快速 cDNA 末端扩增（5' RLM-RACE）是验证 microRNA（miRNA）和类病毒衍生小 RNA（vd-sRNA）对靶基因直接切割的广泛接受的方法。然而，如果切割发生在靶 RNA 的 3' 末端，由于用于 5' RLM RACE 的嵌套 PCR 引物设计的序列长度不足，因此无法使用该方法。为了克服这一障碍，我们开发了 3' RNA 连接酶介导的快速 cDNA 末端扩增（3' RLM RACE）。在该方法中，用具有 5' 腺苷酸化和 3' 封闭的寡核苷酸接头连接到切割 RNA 的 3' 末端，然后使用基因特异性引物进行 PCR 扩增。换句话说，在 3' RLM RACE 中，使用 5' 片段而不是小的 3' 片段来映射 3' 末端。通过检查生物信息学预测和平行分析 RNA 末端（PARE）在感染马铃薯纺锤块茎类病毒的番茄植物中氯离子通道蛋白 CLC-b 样 mRNA 的证实切割位点，验证了这里开发的方法。本研究中开发的 3' RLM RACE 有可能验证 miRNA 和 vd-sRNA 在其 3' 非翻译区（3'UTR）介导的 mRNA 切割。
