Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de Recherche Scientifique (CNRS) UMR 7104 - Institut National de Santé et de Recherche Médicale (Inserm) U1258 - Université de Strasbourg, Illkirch, France.
Methods Mol Biol. 2024;2723:113-123. doi: 10.1007/978-1-0716-3481-3_7.
In eukaryotes, a non-templated poly-adenosine (poly(A)) tail is added co-transcriptionally to almost every messenger RNA (mRNA). The length of this poly(A) tail changes during the lifetime of mRNAs and has been shown in many circumstances to be an important factor controlling transcript fates. Yet, the measure of the length of this homogenous nucleotide sequence is technically challenging, making it difficult to assess its dynamic variation. In this chapter, we describe an RNA-ligation-based RACE-PAT (Rapid Amplification of cDNA End-Poly(A) Tail) assay to monitor the poly(A) tail length of mRNAs. In the first step, an RNA oligonucleotide is ligated to mRNA 3' ends providing an anchoring site to prime cDNA synthesis, avoiding the bias introduced by oligo(dT)-derived primers. Afterward, reverse transcription is performed with an anchor primer with a unique 5' extension. The choice of the oligonucleotide 3' end at this step allows further flexibility to amplify modified tails, for example, by uridylation. Next, short DNA fragments encompassing the poly(A) tails are amplified by Polymerase Chain Reaction (PCR) using as forward primer, a transcript-specific primer hybridizing close to the transcript polyadenylation signal, and as reverse primer, an oligonucleotide corresponding to the 5' extension of the primer used for cDNA synthesis, ensuring that only cDNAs are amplified. The resulting DNA fragments are then visualized after size fractionation by electrophoresis. This method does not provide exact nucleotide count and composition but has the advantage of allowing the processing of many samples in parallel at a low cost.
在真核生物中，几乎每一种信使 RNA（mRNA）都在转录过程中被加上一段非模板化的多腺苷酸（poly(A)）尾巴。这种 poly(A)尾巴的长度在 mRNA 的生命周期中会发生变化，并且在许多情况下已被证明是控制转录命运的重要因素。然而，由于这种均一核苷酸序列的长度测量技术具有挑战性，因此很难评估其动态变化。在本章中，我们描述了一种基于 RNA 连接的 RACE-PAT（快速 cDNA 末端多聚（A）尾扩增）测定法，以监测 mRNAs 的 poly(A)尾巴长度。在第一步中，将 RNA 寡核苷酸连接到 mRNA 3' 末端，为 cDNA 合成提供锚定位点，从而避免了寡聚（dT）衍生引物带来的偏差。之后，使用带有独特 5' 延伸的锚定引物进行反转录。在此步骤中，寡核苷酸 3' 端的选择允许进一步灵活地扩增修饰的尾巴，例如通过尿嘧啶化。接下来，通过聚合酶链反应（PCR）扩增包含 poly(A)尾巴的短 DNA 片段，正向引物是杂交到转录物多聚腺苷酸化信号附近的转录物特异性引物，反向引物是与 cDNA 合成中使用的引物的 5' 延伸相对应的寡核苷酸，以确保仅扩增 cDNA。然后，通过电泳进行大小分离后，可观察到所得 DNA 片段。该方法不能提供确切的核苷酸计数和组成，但具有允许以低成本并行处理许多样品的优点。
