Archibugi Livia, Ruta Veronica, Panzeri Valentina, Redegalli Miriam, Testoni Sabrina Gloria Giulia, Petrone Maria Chiara, Rossi Gemma, Falconi Massimo, Reni Michele, Doglioni Claudio, Sette Claudio, Arcidiacono Paolo Giorgio, Capurso Gabriele
Pancreato-Biliary Endoscopy and Endosonography Division, Pancreas Translational & Clinical Research Center, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
Department of Pathology, San Raffaele Scientific Institute IRCCS-Vita Salute San Raffaele University, 20132 Milan, Italy.
Cells. 2020 Nov 30;9(12):2561. doi: 10.3390/cells9122561.
Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of < 20% of cases. The majority of PDAC patients undergo endoscopic ultrasound (EUS)-guided tissue acquisition (EUS-TA), but RNA has been rarely extracted from EUS-TA with scanty results. Herein, we aimed to determine the best conditions for RNA extraction and analysis from PDAC EUS-TA samples in order to carry out molecular analyses. PDAC cases underwent diagnostic EUS-TA, with needles being a 25G fine needle aspiration (FNA) in all patients and then either a 20G lateral core-trap fine needle biopsy (FNB) or a 25G Franseen FNB; the conservation methods were either snap freezing, RNALater or Trizol. RNA concentration and quality (RNA integrity index; RIN) were analyzed and a panel of genes was investigated for tissue contamination and markers of molecular subtype and aggressivity through qRT-PCR. Seventy-four samples from 37 patients were collected. The median RNA concentration was significantly higher in Trizol samples (10.33 ng/uL) compared with snap frozen (0.64 ng/uL; < 0.0001) and RNALater (0.19 ng/uL; < 0.0001). The RIN was similar between Trizol (5.15) and snap frozen samples (5.85), while for both methods it was higher compared with RNALater (2.7). Among the needles, no substantial difference was seen in terms of RNA concentration and quality. qRT-PCR analyses revealed that samples from all needles were suitable for the detection of PDAC subtype markers (GATA6 and ZEB1) and splice variants associated with mutational status (GAP17) as well as for the detection of contaminating tissue around PDAC cells. This is the first study that specifically investigates the best methodology for RNA extraction from EUS-TA. A higher amount of good quality RNA is obtainable with conservation in Trizol with a clear superiority of neither FNA nor FNB needles. RNA samples from EUS-TA are suitable for transcriptome analysis including the investigation of molecular subtype and splice variants expression.
转录组分析能够区分胰腺导管腺癌（PDAC）的不同亚型，这些亚型具有不同的预后和化疗反应。然而，从胰腺组织中提取RNA很麻烦，且主要是从手术样本中提取，而手术样本仅代表不到20%的病例。大多数PDAC患者接受内镜超声（EUS）引导下的组织获取（EUS-TA），但从EUS-TA中提取RNA的情况很少，结果也很有限。在此，我们旨在确定从PDAC的EUS-TA样本中提取和分析RNA的最佳条件，以便进行分子分析。PDAC病例接受诊断性EUS-TA，所有患者均使用25G细针穿刺抽吸（FNA）针，然后要么使用20G侧向芯吸式细针活检（FNB）针，要么使用25G Franseen FNB针；保存方法为速冻、RNA Later或Trizol。分析RNA浓度和质量（RNA完整性指数；RIN），并通过qRT-PCR研究一组基因，以检测组织污染以及分子亚型和侵袭性的标志物。收集了37例患者的74个样本。与速冻样本（0.64 ng/μL；<0.0001）和RNA Later样本（0.19 ng/μL；<0.0001）相比，Trizol样本中的RNA浓度中位数显著更高（10.33 ng/μL）。Trizol样本（5.15）和速冻样本（5.85）的RIN相似，而这两种方法的RIN均高于RNA Later样本（2.7）。在不同的针之间，RNA浓度和质量方面未见实质性差异。qRT-PCR分析显示，所有针获取的样本均适用于检测PDAC亚型标志物（GATA6和ZEB1）以及与突变状态相关的剪接变体（GAP17），也适用于检测PDAC细胞周围的污染组织。这是第一项专门研究从EUS-TA中提取RNA的最佳方法的研究。使用Trizol保存可获得更高质量的RNA，且FNA针和FNB针均无明显优势。EUS-TA获取的RNA样本适用于转录组分析，包括分子亚型和剪接变体表达的研究。
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