Bovinder Ylitalo Erik, Vidman Linda, Harlid Sophia, Van Guelpen Bethany
Department of Diagnostics and Intervention, Oncology, Umeå University, Umeå, Sweden.
Wallenberg Centre for Molecular Medicine, Umeå University, Umeå, Sweden.
PLoS One. 2025 Mar 19;20(3):e0318834. doi: 10.1371/journal.pone.0318834. eCollection 2025.
Transcriptomics is an important OMICs method that is often unavailable in biobank research. Frozen blood samples are routinely collected and stored in medical biobanks, but transcriptional studies have been limited due to technical difficulties of extracting high-quality RNA from blood frozen in standard tubes (without RNA preservatives). We aimed to determine whether biobanked buffy coat samples stored at -80°C for up to 23 years could be successfully used for mRNA sequencing. We used a CryoXtract CXT 350 to remove frozen sample cores, which were immersed in RNA preservative during thawing prior to RNA extraction. RNA sequencing was then performed on extractions from pooled samples as well as from 23 buffy coat samples from prospective colorectal cancer cases and 23 matched controls included in the population-based, prospective Northern Sweden Health and Disease Study (NSHDS). For all samples, two library preparation methods were used (Illumina TruSeq Stranded mRNA poly-A selection and Illumina Stranded Total RNA with Ribo-Zero Globin). RNA yields of over 1 µg were obtained from the majority of NSHDS samples (mean = 2.57 µg), and over 92% of samples had RIN values of ≥ 6, indicating suitability for downstream analyses. In conclusion, we developed a method for successfully extracting and sequencing high-quality mRNA from frozen buffy coat samples stored long term in tubes with no RNA preservative.
转录组学是一种重要的组学方法，在生物样本库研究中常常无法使用。冷冻血液样本通常会被收集并储存在医学生物样本库中，但由于从标准试管（不含RNA防腐剂）中冷冻的血液中提取高质量RNA存在技术困难，转录研究一直受到限制。我们旨在确定储存在-80°C长达23年的生物样本库中的血沉棕黄层样本是否能成功用于mRNA测序。我们使用CryoXtract CXT 350取出冷冻样本芯，在解冻期间将其浸入RNA防腐剂中，然后进行RNA提取。随后对来自合并样本以及来自基于人群的前瞻性瑞典北部健康与疾病研究（NSHDS）中23例前瞻性结直肠癌病例和23例匹配对照的血沉棕黄层样本提取物进行RNA测序。对于所有样本，使用了两种文库制备方法（Illumina TruSeq链特异性mRNA多聚腺苷酸选择法和Illumina链特异性总RNA去除珠蛋白法）。大多数NSHDS样本（平均值 = 2.57 µg）获得了超过1 µg的RNA产量，超过92%的样本RIN值≥6，表明适合进行下游分析。总之，我们开发了一种从长期保存在无RNA防腐剂试管中的冷冻血沉棕黄层样本中成功提取和测序高质量mRNA的方法。
