The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K2EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

background or methods

https://www.mdpi.com/2072-6694/11/4/458/pdf

The extracellular RNA quality control (exRNAQC) study : testing and controlling pre-analytical variables of RNA sequencing on liquid biopsies

Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol.

LncRNAs are defined as RNA transcripts greater than 200 nucleotides in length that have no or limited protein-coding potential. Basal expression of lncRNAs appeared important for various homeostatic processes, like gene imprinting cell differentiation and organogenesis. Moreover, it has been demonstrated that lncRNAs play an important role in tumorigenesis and metastasis. Some lncRNAs were stably detected in exosomes, which are widely found in body fluids. Several studies validated the use of exosomal lncRNAs as minimally invasive diagnostic and prognostic markers in several types of cancers. In addition, exosomal lncRNAs have been associated with drug resistance of tumor cells, suggesting a clinical application in cancer-targeted therapy. Despite the recent increase of studies on exosomal lncRNAs, their clinical significance in cancer diagnosis, prognosis and treatment needs to be fully explored. The methodologies for their detection with high purity and accuracy must be also improved in order to implement their use in clinical routine. This review aims to summarize the main recent technologies available for the isolation of exosomal lncRNAs, their status as a liquid biopsy as well as their future perspectives.

methods

/pdf/exosomal-lncrnas-the-newest-promising-liquid-biopsy-3t4nghauud.pdf

The extracellular RNA quality control (exRNAQC) study : testing and controlling pre-analytical variables of RNA sequencing on liquid biopsies

Citations

Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol.

Exosomal lncRNAs: the newest promising liquid biopsy

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