Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Lab Invest. 2020 Oct;100(10):1345-1355. doi: 10.1038/s41374-020-0446-z. Epub 2020 May 28.
This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3' massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.
本研究旨在比较标准 RNA 测序（RNA-Seq）和 3' 大规模 cDNA 末端分析（MACE）RNA-Seq 用于分析新鲜组织的潜力，并描述通过 MACE 对福尔马林固定石蜡包埋（FFPE）存档人类样本进行的转录组分析。为了在新鲜组织上将 MACE 与标准 RNA-Seq 进行比较，从四位接受玻璃体视网膜手术的患者中采集了四个健康的结膜，将其对半切开并立即转移到 RNA 裂解缓冲液中，而无需事先固定，然后分别进行标准 RNA-Seq 或 MACE RNA-Seq 分析。为了评估 FFPE 制备对 MACE 的影响，第三部分在福尔马林中固定并进行石蜡包埋处理，然后将其转录谱与未经固定的标本进行比较，并用 MACE 进行分析。为了研究 FFPE 储存时间对 MACE 结果的影响，还分析了 24 例患者的 24 个 FFPE 处理的结膜样本。通过 MACE 和标准 RNA-Seq 在新鲜组织上共检测到 19659 个转录基因，而通过 MACE 或 RNA-Seq 分别特异性检测到 3251 和 2213 个转录本。尽管进行了归一化处理，但标准 RNA-Seq 产生的转录本往往比 MACE 技术更长，这表明 MACE 技术不太容易受到长度偏倚的影响。FFPE 处理对 MACE 测序结果的影响可以忽略不计。几项质量控制测量表明，在石蜡中长期储存不会降低 MACE 文库的多样性。我们注意到，在石蜡中储存时间与原始读数数量之间存在非线性关系，在前 1000 天内呈加速下降，而在较旧的样本中，数量则相对稳定。有趣的是，检测到的转录基因数量与 FFPE 储存时间无关。从 FFPE 样本中可以提取出足够质量和数量的 RNA，使用 MACE 技术获得全面的转录组分析。因此，我们提出 MACE 是利用组织学存档中 FFPE 样本的新机会。
