Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

Purpose: To determine the optimal RNA extraction and library generation protocols for mouse hippocampal tissue acquired by laser capture microdissection (LCM). Methods: Hippocampal subregion CA2 was captured from eight micron fresh frozen brain sections from AMIGO2 EGFP mice. Total RNA was extracted using the PicoPure (LCM standard) and QIAGEN micro RNeasy RNA extraction kits. RNA quantity and quality was assessed using a Bioanalzyer. Resulting RNA was used to generate cDNA libraries using NuGEN or SMARTer low input RNA-Seq library kits. We compared the effects of RNA quality and library generation approach to determine the methods that detected the greatest number of genes/exons with even coverage using minimal rounds of PCR. Results: We determined that the QIAGEN RNA extraction kit resulted in far superior RNA compared to the Picopure RNA extraction kit. We also found the NuGEN library generation kit that depletes ribosomal RNA towards the end of the protocol, led to higher cDNA library yields that required fewer rounds of PCR while providing even gene coverage and detection. Overall design: RNA from laser-captured tissue from mouse hippocampus was extracted using two low input methods generating a lower quality RNA sample (RIN 7, PicoPure) and higher quality sample (RIN 9, QIAGEN). Each sample was used to prepare cDNA using two commerically available low-input library generation methods (Clontech or NuGEN). The effect of RNA quality and library generation method were compared. The effect of shearing cDNA and PCR amplification were also tested for libraries made with the QIAGEN extracted RNA and NuGEN library kits (3 libraries, 16, 18 or 20 cycles of PCR). The libraries were multiplexed and run on the Illumina NextSeq500 instrument.

研究目的：本研究旨在确定适用于激光捕获显微切割（Laser Capture Microdissection, LCM）获取的小鼠海马组织的最优RNA提取与文库构建方案。
实验方法：从AMIGO2 EGFP小鼠的8μm新鲜冷冻脑切片中捕获海马亚区CA2；分别采用PicoPure（LCM标准试剂盒）与QIAGEN微量RNeasy RNA提取试剂盒提取总RNA；通过生物分析仪（Bioanalyzer）评估RNA的产量与质量；随后利用NuGEN或SMARTer低起始量RNA测序文库构建试剂盒，以提取得到的RNA制备cDNA文库。本研究比较了RNA质量与文库构建策略的影响，旨在筛选出仅需少量PCR循环即可实现均匀覆盖并检测最多基因/外显子的实验方法。
实验结果：相较于PicoPure RNA提取试剂盒，QIAGEN RNA提取试剂盒获取的RNA质量显著更优；同时发现，在实验流程后期进行核糖体RNA去除的NuGEN文库构建试剂盒，可获得更高的cDNA文库产量，所需PCR循环数更少，且基因覆盖度均匀、检测效果更佳。
整体实验设计：本研究采用两种低起始量RNA提取方法，从激光捕获的小鼠海马组织中分别获得RNA完整性指数（RNA Integrity Number, RIN）为7的低质量RNA样本（PicoPure提取）与RIN为9的高质量RNA样本（QIAGEN提取）；分别使用两款商业化低起始量文库构建试剂盒（Clontech与NuGEN）对每种RNA样本制备cDNA文库，以比较RNA质量与文库构建方法的影响。此外，针对使用QIAGEN提取的RNA与NuGEN文库试剂盒构建的文库，本研究还测试了cDNA片段化与PCR扩增的效果，共设置3组文库，分别采用16、18或20轮PCR循环。所有文库经多重测序后，在Illumina NextSeq500测序仪上完成测序。