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Objectives: The small RNAs of bovine plasma and whole blood were analysed using next-generation sequencing to quantify, profile and compare the microRNAs (miRNA) and piRNA signatures in both bio fluids. Results: Evaluating read-count data resulted in a proportion of 5.0 ± 2.9 % of miRNAs in plasma while 38.2 ± 3.4 % were identified in whole blood. Regarding piRNAs, the percentages in both matrices were nearly the same: 1.4 ± 0.8 % of piRNAs in plasma and 1.9 ± 0.8 % in whole blood. Investigation of the ten most abundant miRNAs and piRNAs in both bio fluids revealed that two miRNAs and seven piRNAs were identical. Comparing the read-count values of these matching pairs highlighted that miRNA and piRNA levels in blood exceeded the abundance of their corresponding miRNAs and piRNAs in plasma, except liver-specific miR-122 and three piRNAs. Conclusions: The data strengthened evidence that the circulating small RNA signature in plasma is not only influenced by hematocytes and certain small RNAs could originate from other sources than cellular blood components. [ABSTRACT FROM AUTHOR]

There are several protocols and kits for the extraction of circulating RNAs from plasma with a following quantification of specific genes via RT-qPCR. Due to the marginal amount of cell-free RNA in plasma samples, the total RNA yield is insufficient to perform Next-Generation Sequencing (NGS), the state-of-the-art technology in massive parallel sequencing that enables a comprehensive characterization of the whole transcriptome. Screening the transcriptome for biomarker signatures accelerates progress in biomarker profiling for molecular diagnostics, early disease detection or food safety. Therefore, the aim was to optimize a method that enables the extraction of sufficient amounts of total RNA from bovine plasma to generate good-quality small RNA Sequencing (small RNA-Seq) data. An increased volume of plasma (9 ml) was processed using the Qiagen miRNeasy Serum/Plasma Kit in combination with the QIAvac24 Plus system, a vacuum manifold that enables handling of high volumes during RNA isolation. 35 ng of total RNA were passed on to cDNA library preparation followed by small RNA high-throughput sequencing analysis on the Illumina HiSeq2000 platform. Raw sequencing reads were processed by a data analysis pipeline using different free software solutions. Seq-data was trimmed, quality checked, gradually selected for miRNAs/piRNAs and aligned to small RNA reference annotation indexes. Mapping to human reference indexes resulted in 4.8±2.8% of mature miRNAs and 1.4±0.8% of piRNAs and of 5.0±2.9% of mature miRNAs for bos taurus. [ABSTRACT FROM AUTHOR]