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. 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：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新鲜冷冻脑组织切片中捕获海马亚区CA2。分别采用PicoPure（LCM标准方案）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的浓度与质量。所得RNA分别使用NuGEN或SMARTer低起始量RNA测序文库构建试剂盒制备cDNA文库。本研究对比了RNA质量与文库构建方式的影响，旨在筛选出在最少PCR循环数下，可实现最均匀覆盖度并检测到最多基因/外显子的实验方法。
实验结果：本研究发现，QIAGEN RNA提取试剂盒所得RNA的质量远优于PicoPure试剂盒。同时，在实验流程后期进行核糖体RNA消减的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，且能实现更均匀的基因覆盖度与检测效果。本研究通过两种低起始量RNA提取方法，从小鼠海马激光捕获组织中分别获得了低质量RNA样品（RIN值7，对应PicoPure提取组）与高质量RNA样品（RIN值9，对应QIAGEN提取组）。分别使用两种商业化低起始量文库构建试剂盒（Clontech与NuGEN）对各样品制备cDNA文库，对比了RNA质量与文库构建方式的影响。此外，针对使用QIAGEN提取的RNA与NuGEN文库试剂盒构建的3个文库（分别采用16、18或20个PCR循环），本研究还测试了cDNA片段化与PCR扩增的影响。所有文库经多重测序后，在Illumina NextSeq500测序仪上完成上机测序。