Micro RNA Sequencing in Circulating Tumor Cells

MicroRNAs (miRNAs) play a crucial role in the analysis of circulating tumor cells (CTCs) due to their unique properties and involvement in cancer biology. miRNAs in CTCs can help to identify early changes associated with metastasis, allowing for early intervention. Specific miRNAs are associated with drug resistance mechanisms, and their presence or absence in CTCs can help predict the effectiveness of treatment options, enabling personalized therapy adjustments. Changes in miRNA expression levels in CTCs can also be used to monitor disease progression or detect early signs of recurrence after treatment. This is especially important in managing cancers that are prone to relapse. Therefore, the in-depth characterization of physiological miRNA functions is an important basic research question, and miRNAs even have a high potential as biomarkers both for prognosis and diagnosis. To exploit this potential, it is mandatory to accurately quantify the miRNA expression not only in tumor bulk, but also on the single CTC level. Here, we describe a protocol that facilitates miRNA sequencing library preparation in CTCs. The protocol can be combined with different single-cell isolation methods (e.g., micromanipulation and FACS sorting). After cell lysis, sequencing adapters are ligated to the miRNAs, other ncRNA species and adapter dimers are reduced by exonuclease digest, the miRNA library is reverse transcribed, amplified and purified. Furthermore, quality controls are described to select only high-quality samples for sequencing.