sample type: serum from patient with neuro endocrine tumor
Treatment protocol
n/a
Growth protocol
Samples were extracted at the Department of Pathology, Erasmus MC, Rotterdam, the Netherlands. Use of tissue samples for scientific reasons was approved by an institutional review board (MEC 02.981 and CCR2041). Samples were used according to the “Code for Proper Secondary Use of Human Tissue in The Netherlands” developed by the Dutch Federation of Medical Scientific Societies (FMWV (Version 2002, update 2011).
Extracted molecule
total RNA
Extraction protocol
miRNAs were purified from serum sample using TaqMan® ABC Purification Kit - Human Panel A and B (Life Technologies, PN 4473087 and 4473088). All reagents are provided in the kit. These panels consist of superparmagnetic Dynabeads® covalently bound to a unique set of ~380 anti-miRNA oligonucleotides for each panel. The miRNAs in each panel match the miRNAs in Megaplex Pools A and B described below. Each panel includes exogenous and endogenous controls. Briefly, for each panel 100ul of lysis buffer was added to 50 µl of serum. 2 µl of 1nM an external control (ath-miR-159a) was added to monitor the extraction process, followed by the addition of 80 µl of beads ( 80 X 10^6 beads). The tubes were shaken at 1200 rpm at 30˚C for 40 minutes then washed three times with wash buffer using a magnetic bead separator. The bound miRNAs were eluted from the beads with 100 µl elution buffer and incubation at 70˚C for 3 minutes. The eluted miRNA was stored at -80˚C until ready to use.
Label
FAM
Label protocol
For miRNA profiling, Megaplex™ Primer Pools A and B v3.0 (PN 4444750) were used in conjunction with their matching TaqMan® MicroRNA Array Cards (PN 4444913). Each Pool contains either reverse transcription primers or preamplification primers that target 377 unique miRNAs and additional control targets and allows for profiling up to of 754 unique miRNAs across the two pools. Each sample was run separately with Pool A and B according to a modified protocol for blood plasma outlined in the Life Technologies Application Note: “Optimized blood plasma protocol for profiling human miRNAs using the OpenArray® Real-Time PCR System”, 2011, PN 4399721 rev C. Briefly, 3 µl of of the miRNA sample that was isolated with the ABC kit was reverse transcribed with the Megaplex™ RT Primer Pool A or B in a 10 µl final volume. The RT reaction was thermal cycled (2 min at 16˚C, 1 min at 42˚C, 1 sec at 50˚C, for 40 cycles) and the enzyme inactivated at 85˚C for 5 min. The entire RT reaction was combined with its matching Megaplex™ PreAmp Primer Pool and TaqMan® PreAmp Master Mix (PN 4391128) in a final volume of 50 µl. Preamplification was run using the following cycling conditions: 10 min at 95˚C; 2 min at 55˚C; 2 min at 72˚C; 15s at 95˚C, 4 min at 60˚C for 14 cycles; 99˚C for 10 min. The final preamplification product was diluted 1:100 in 1X TaqMan® Universal Master Mix II then loaded onto the matching TaqMan® MicroRNA Array Card and run on a QuantStudio™ 12K Flex System fitted with a TaqMan® Array Card Block under universal cycling. Each sample was run on Card A and Card B.
Hybridization protocol
n/a
Scan protocol
n/a
Data processing
Raw data files (.eds) were imported and analyzed in using ExpressionSuite v1.0.4 (Life Technologies), a software data analysis tool that can easily import and analyze large raw data files. In these experiments the Cq is defined as the fractional cycle at which the amplification plot crosses the fluorescence threshold (Ct). The baseline was set automatically and the threshold was manually set at 0.2. For gene expression analysis a Ct cutoff that provides reliable results for low expressing targets is identified. The cutoff will depend on the platform, number of replicates (technical and biological) and number of preamplification cycles if preamplification was included. For TaqMan Array cards without preamplification, a Ct <30 is generally considered positive and indicates expression; Ct between 30 – 32 are considered low but detectable. Ct between 32-40 represent very low expression. A larger number of replicates or low standard deviation of Ct increases the confidence and power of results in this range. To capture as many differentially expressed miRNAs as possible the threshold was set at 40 and statistical analysis was applied to identify the most relevant and consistent (i.e. significant) results. Undetermined values were replaced with the maximal number of cycles (40). Non-human RNAs were removed from the dataset before normalization. Each assay was treated as an independent target. Ct values were normalized using global normalization according to [1]. RNU44, RNU48 and U6.snRNA were removed from the dataset before further analysis because these have been shown to be absent in serum [2]. Batch effects were corrected using Combat as implemented in the sva Bioconductor package (v3.10.0). 1. Mestdagh, P., et al., A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol, 2009. 10(6): p. R64. 2. Gillis, A.J., et al., Targeted serum miRNA (TSmiR) test for diagnosis and follow-up of (testicular) germ cell cancer patients: a proof of principle. Mol Oncol, 2013. 7(6): p. 1083-92.
