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从 COMET 生物样本库人类代谢组织样本中优化 RNA 提取方法。

Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank.

机构信息

Biocommunication in Cardio-Metabolism (BC2M), University of Montpellier, 15 avenue Charles Flahault, 34093, Montpellier Cedex 5, France.

RD Néphrologie, 2 rue des Muriers, 34090, Montpellier, France.

出版信息

Sci Rep. 2021 Oct 25;11(1):20975. doi: 10.1038/s41598-021-00355-x.

DOI:10.1038/s41598-021-00355-x
PMID:34697345
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8545963/
Abstract

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

摘要

生物银行的组织构建需要小心处理和储存生物材料，以保证样本的质量。组织准备对于进一步的应用也很关键，如转录组谱分析。在这项研究中，我们的目的是评估不同的破坏技术（FastPrep-24 仪器、GentleMACS 分离机和注射器/针头）和匀浆缓冲液（RLT 与 QIAzol）对 COMET 生物银行中代谢组织（脂肪组织、肝脏和骨骼肌）的 RNA 纯度和质量的影响。对于使用的所有匀浆方法和组织类型，A260/280 比值达到了≥1.8，这在人类组织和细胞系中是常见的范围，而 A260/230 比值则低于 1.8，其中最低值是用 GentleMACS 分离机获得的。此外，GentleMACS 分离机与 QIAzol 试剂结合使用，除了肌肉组织外，对所有测试的组织都给出了最高的 RIN 值和 28S/18S 比值。进行 RT-qPCR 时，不同管家基因的 Ct 值可能会受到提取方法和样本 RNA 质量的影响。总之，我们已经证明，不同的破坏技术和匀浆缓冲液会影响 RNA 的纯度和一些质量标志物，并且还会影响代谢组织中 mRNA 的 RT-qPCR 定量。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/5ee8d8d58ba7/41598_2021_355_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/b0ac70c64c68/41598_2021_355_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/7730b6006dc6/41598_2021_355_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/3ca1912e7e55/41598_2021_355_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/20a79558f313/41598_2021_355_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/ee79ba558c64/41598_2021_355_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/5ee8d8d58ba7/41598_2021_355_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/b0ac70c64c68/41598_2021_355_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/7730b6006dc6/41598_2021_355_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/3ca1912e7e55/41598_2021_355_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/20a79558f313/41598_2021_355_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/ee79ba558c64/41598_2021_355_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a123/8545963/5ee8d8d58ba7/41598_2021_355_Fig6_HTML.jpg

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