Can milk cell or skim milk miRNAs be used as biomarkers for early pregnancy detection in cattle?

<div><p>The most critical phase of pregnancy is the first three weeks following insemination. During this period about 50% of high yielding lactating cows suffer embryonic loss prior to implantation, which poses a high economic burden on dairy farmers. Early diagnosis of pregnancy in cattle is therefore essential for monitoring breeding outcome and efficient production intervals. Regulated microRNAs (miRNAs) that reach easily accessible body fluids via a ‘liquid biopsy’ could be a new class of pregnancy predicting biomarkers. As milk is obtained regularly twice daily and non-invasively from the animal, it represents an ideal sample material. Our aim was to establish a pregnancy test system based on the discovery of small RNA biomarkers derived from the bovine milk cellular fraction and skim milk of cows. Milk samples were taken on days 4, 12 and 18 of cyclic cows and after artificial insemination, respectively, of the same animals (n = 6). miRNAs were analysed using small RNA sequencing (small RNA Seq). The miRNA profiles of milk cells and skim milk displayed similar profiles despite the presence of immune cell related miRNAs in milk cells. Trends in regulation of miRNAs between the oestrous cycle and pregnancy were found in miR-cluster 25~106b and its paralog cluster 17~92, miR-125 family, miR-200 family, miR-29 family, miR-15a, miR-21, miR-26b, miR-100, miR-140, 193a-5p, miR-221, miR-223, miR-320a, miR-652, miR-2898 and let-7i. A separation of cyclic and pregnant animals was achieved in a principal component analysis. Bta-miRs-29b, -221, -125b and -200b were successfully technically validated using quantitative real-time PCR, however biological validation failed. Therefore we cannot recommend the diagnostic use of these miRNAs in milk as biomarkers for detection of bovine pregnancy for now.</p></div

Foldchanges/ratios of regulated miRNA during oestrous cycle (cy) and pregnancy (p) measured in RT-qPCR.

<p>Foldchanges/ratios of regulated miRNA during oestrous cycle (cy) and pregnancy (p) measured in RT-qPCR.</p

Comparison of MC and SM miRNA profile.

<p>A) Correlation between miRNA mean values of all MC and SM NGS reads. Normalized NGS reads were used for calculation of Pearson Correlation Coefficient B) Heatmap of the top 100 miRNAs. Data was normalized, rld transformed and clustered using DESeq2 C) PCA showing MC and SM specific clustering. Clustering was performed using DESeq2.</p

log2 fold-change of selected miRNAs in the milk cellular and skim milk fraction measured with high throughput sequencing.

<p>log2 fold-change of selected miRNAs in the milk cellular and skim milk fraction (n = 6, except SM pregnant day 18 n = 5) measured with high throughput sequencing. All fold-changes and p-values for pregnant/cyclic data were calculated using Deseq2. p-values between cyclic (18c/4c) and pregnant Day 18/Day 4 ratios (18p/4p) were calculated using paired t-test on log2 fold-changes *: p<0.05, **: p< 0.01. Error bars are shown as log fold standard error. A) log2 fold-change between pregnancy and oestrous cycle on days 4, 12 and 18 B) log2 fold-change between Day 18 and Day 4 during oestrous cycle and pregnancy. C) PCA of log2 fold-change of the miRNAs: bta-miR-25, bta-miR-93, bta-miR-106b, bta-miR-125b, bta-miR-193a-5p, bta-miR-200b, bta-miR-200c, bta-miR-221, bta-miR-2898, bta-let-7i between days 4 and 18 (Day 18/Day 4) showing the separation of cyclic and pregnant animals.</p

Statistically significant differences in miRNA levels between milk cells and skim milk.

<p>Statistically significant differences in miRNA levels between milk cells and skim milk.</p

Fold-change/ratio of regulated miRNA during oestrous cycle (cy) and pregnancy (p) measured in NGS.

<p>Fold-change/ratio of regulated miRNA during oestrous cycle (cy) and pregnancy (p) measured in NGS.</p

Correlation of miRNA expression between milk cells and skim milk.

<p>Correlation of miRNA expression between milk cells and skim milk.</p

Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing

<div><p>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 <i>bos taurus</i>.</p></div

Percentage of mapped and annotated miRNA/piRNA reads compared to the total number of sequences.

<p>Percentage of mapped and annotated miRNA/piRNA reads compared to the total number of sequences.</p

Sequence motif analysis to evaluate piRNA 5′-T-bias.

<p>Image [A] shows the piRNA reference that was used for alignment and Image [B] represents the nucleotide composition of mapped piRNAs pointing out the T-bias at 5′.</p