Isolating high-quality RNA for RNA-Seq from 10-year-old blood samples

Abstract

There is much interest in analysing RNA, particularly with RNA Sequencing, across both research and diagnostic domains. However, its inherent instability renders it susceptible to degradation. Given the imperative for RNA integrity in such applications, proper storage and biobanking of blood samples and successful subsequent RNA isolation is essential to guarantee optimal integrity for downstream analyses. Especially for larger collections, it would be particularly beneficial if these methods would additionally offer affordability, minimal blood volume requirements and also long-term storage. In this study, RNA of high quality, suitable for transcriptomics, has been successfully isolated from 400 µL of EDTA and citrated whole blood samples in Boom's lysis buffer stored at -85 °C for 10 years. Isolation was carried out using a modified Zymo Research Quick-RNA kit protocol. This isolation method showed significant improvement in RNA integrity when compared to RNA extracted using the original Boom method. RNA Sequencing provided high-quality data comparable to that of other studies using recently frozen blood in RNA stabilisation tubes. Additionally, sequencing data from blood collected in citrate and EDTA anticoagulants also showed excellent correlation.

Keywords: Biobanking; Long-term storage; Multi-omics; RNA isolation; RNA-Seq; Transcriptomics.

Fig. 1

Percentages of exonic, intronic and intergenic regions in EDTA and citrated samples. Distribution of reads to genomic regions shows a low proportion of reads from intergenic regions, excluding major DNA contamination in both EDTA and citrated samples.

Fig. 2

Good correlation in the median gene expression in Transcript per Million (TPM) was observed between the RNA-Seq data collected from RNA isolated from 10-year-old blood samples and data from healthy controls frozen for 1–2 years from the Kopp public dataset.

Fig. 3

Good correlation in the median gene expression was observed between data from EDTA and citrated whole blood.

Fig. 4

Comparison plot (produced with NOISeq v.2.40^,) showing the percentage of each biotype in the genome detected from samples extracted with EDTA (blue) and citrated whole blood (pink). The black line displays the percentage abundance of each biotype within the genome. Abbreviations described in http://feb2021.archive.ensembl.org/info/genome/genebuild/biotypes.html.

Fig. 5

Summary of the modified protocol of the Zymo Research Quick-RNA Whole Blood kit.