ac5b02043_si_002.xlsx (71.97 kB)
Influence of Next-Generation Sequencing and Storage Conditions on miRNA Patterns Generated from PAXgene Blood
dataset
posted on 2015-09-01, 00:00 authored by Christina Backes, Petra Leidinger, Gabriela Altmann, Maximilian Wuerstle, Benjamin Meder, Valentina Galata, Sabine
C. Mueller, Daniel Sickert, Cord Stähler, Eckart Meese, Andreas KellerWhole
blood derived miRNA signatures determined by Next-Generation
Sequencing (NGS) offer themselves as future minimally invasive biomarkers
for various human diseases. The PAXgene system is a commonly used
blood storage system for miRNA analysis. Central to all miRNA analyses
that aim to identify disease specific miRNA signatures, is the question
of stability and variability of the miRNA profiles that are generated
by NGS. We characterized the influence of five different conditions
on the genome wide miRNA expression pattern of human blood isolated
in PAXgene RNA tubes. In detail, we analyzed 15 miRNomes from three
individuals. The blood was subjected to different numbers of freeze/thaw
cycles and analyzed for the influence of storage at −80 or
8 °C. We also determined the influence of blood collection and
NGS preparations on the miRNA pattern isolated from a single individual,
which has been sequenced 10 times. Here, five PAXGene tubes were consecutively
collected that have been split in two replicates, representing two
experimental batches. All samples were analyzed by Illumina NGS. For
each sample, approximately 20 million NGS reads have been generated.
Hierarchical clustering and Principal Component Analysis (PCA) showed
an influence of the different conditions on the miRNA patterns. The
effects of the different conditions on miRNA abundance are, however,
smaller than the differences that are due to interindividual variability.
We also found evidence for an influence of the NGS measurement on
the miRNA pattern. Specifically, hsa-miR-1271-5p and hsa-miR-182-5p
showed coefficients of variation above 100% indicating a strong influence
of the NGS protocol on the abundance of these miRNAs.