Despite rapid progress in the research field of miRNA, consensus regarding optimal quantification methods is still lacking. Therefore, we performed several optimization steps in order to get valuable results from miRNA analysis. miRNA quantification is divided into the following parts: heamolysis assessment, miRNA isolation, RT-qPCR – identification of miRNA, analysis of results. Each part will be commented on separately.
5.17.1 Haemolysis
Recent studies have shown that haemolysis has impact on miRNA content in plasma/serum [287-289] because red blood cells produce significant amounts of miRNA that could be released into plasma after haemolysis. Therefore, firstly, haemolysis was measured spectroscopically using the Harboe calculation: Hb (g/l) = (k[167·2×A415 − 83·6 × A380 − 83·6 × A450]) ÷ 1000 [290]. The Harboe method measures the optical density (415 nm) of oxyhaemoglobin in a sample with additional background correction of the optical densities (380 and 450 nm) [290]. Secondly, haemolysis can be detected by the expression of two miRNA controls (has-miR-451 and has-miR-23a). Has-mir-451 is produced by red-blood cells, while hsa-miR-23a levels are not influenced. These two miRNA species cannot be used as a reference genes in subsequent analyses. Based on these haemolytic controls, we excluded all samples possibly affected by haemolysis.
5.17.2 miRNA isolation
The miRNA isolation was performed using mirVanaTMPARISTMKit. The procedure was performed according to the manufacturer's instructions. Briefly, 250 µl of body fluids (serum/plasma, LAT fluid) were used. The thawed samples were incubated in denaturing solution (2x) for 5-10 minutes at room temperature. Then Acid-Phenol solution supplemented with chloroform was added to samples, vortexed for 60 s and incubated for 15 min. After incubation, the samples were centrifuged for 10 min at maximum speed (12 000 RPM at 4°C). After the centrifugation step, samples were separated into two phases.
The aqueous (upper) phase was carefully collected without disturbing the lower phase. 100
% ethanol was added to the collected aqueous phase and then the samples were thoroughly
69 mixed and transferred into filter cartridge and centrifuged for 1 min at maximum speed (12 000 G) at room temperature. Then, the bound miRNA was washed with miRNA Wash Solution 1 (700 ul) 1 min at maximum speed (12 000 G) at room temperature and Wash solution 2/3 (500 ul) in the same centrifugation settings. This step was repeated twice. After discarding the flow-through samples were centrifuged for 1 min at maximum speed (12 000 G) to remove residual fluid from the filter. RNA was eluted in RNase-free dH2O (50 ul) preheated to 95 ° C. Columns were centrifuged for 1 min at maximum speed (12 000 RPM).
This step was repeated twice. The eluate was collected and stored at -80°C.
5.17.3 RT-qPCR – identification of miRNA
The miRNA detection was performed using miRCURY LNA RT kit and miRCURY LNATM SYBR Green PCR kit containing miRCURYLNATM Focus panels with predefined 179 human miRNAs previously detected in serum/plasma samples (provided by Qiagen). LNA (lock nucleis acids) technology improves binding affinity with high sensitivity and specificity to low expressed miRNAs with improvement of mismatch discrimination.The reverse transcription was processed according to the manufacturer's instructions with small modifications. MiRNA samples were diluted two times with dH20 prior to reverse transcription to limit the concentration of inhibitors during the reaction. The diluted RNA was reverse transcribed in the mix containing 5x miRCURY RT buffer, 10x miRCURX RT enzyme mix, and UNISP6 RNA spike. cDNA was diluted 1:10 in dH2O prior to the qPCR reaction. qPCR reaction consisted from cDNA (2 ul) mixed together with SYBR green mastermix (5.05 ul) containing ROX dye and dH2O (2.45 ul). qPCR was performed on the ABI 7500 Fast instrument.
5.17.4 Analysis of results
Expression levels based on Ct numbers were analysed by Gene Globe online software (Qiagen) and GenEx software 7.0 (MultiD). All miRNAs with Ct greater than 35 were excluded.
70 Quality Control (QC)
UNISP6 was used as control of cDNA synthesis quality [291]. UNISP3 is a spike that was used during the qPCR reaction as control of PCR efficiency and for run to run normalization.
Importantly, spikes were not used as normalisator of gene expression analysis.
Normalization and reference gene
One of the main challenges in miRNA analysis is proper normalization, which limits the effects of technical variability. The global normalization procedure, using the global mean as a normalizer, is frequently used for miRNA measurement in the absence of a stable reference gene. On the other hand, the NormFinder algorithm is based on the selection of 1 or more endogenous controls with stable expression across all conditions in experimental groups. In our analysis, four different normalization analyses were performed.
1. Normfinder algorithm I. (reference genes for normalization were selected automatically)
2. Normfinder algorithm II. (5 selected genes: hsa-mir-15b-5p, hsa-mir-23b-3p, hsa- mir-24-3p, hsa-mir-222-3p)
3. Genorm normalization ( selected genes: hsa-mir-17-5p and hsa-mir-106a-5p) 4. Global normalization (global Ct averaging of all miRNAs)
Prediction of miRNA target genes
The accurate prediction of miRNA targets is the next important step in the interpretation of the results of miRNA analysis. Today, several online databases, which are freely available, represent valuable tools to identify possible target genes of miRNA. The Rnacentral database (https://rnacentral.org/) characterizes miRNA with recent research outputs. Mirbase (https://mirbase.org/) acts as a repository of miRNA sequences and annotations. The target genes of our selected miRNAs were predicted using the free online miRBD database (http://mirdb.org/). miRBD database is an online tool for miRNA target prediction and functional annotations, which it possesses newest algorithm system [292]. The selection of miRNA target genes was performed based on our own pipeline. First, we select target genes with an intended target score of 50 for each miRNA with altered expression. We selected
71 the target genes with the highest frequency of occurrence across all the altered miRNAs. The implication of these selected genes in processes related to adipogenesis or angiogenesis was evaluated based on the available literature. This pipeline of analysis was applied on more than 2000 genes.
5.18 Methods used during internship in Jeltsch laboratory, University of Helsinki The purification of the inactive form of VEGF-C (pro-VEGF-C) with its activation by thermolysin protease together with the Ba/F3 viability assay represent procedures performed during my internship with Dr. Jeltsch, University of Helsinki, Finland. Chemicals, peptides, and recombinant proteins used during the internship were not included in the tables of supplements
5.18.1 Purification of pro-VEGF-C
The recombinant protein was purified from insect cells (S2 cells) expressing human pro- VEGF-C with polyhistidine chains in dimeric and monomeric forms. The secretion of protein in the media was induced by CuSO4 (50mM). The first step of purification is based on His-tag purification. To capture His-tag protein we used Ni2+ resin (Nitrilotriacetic acid coupled with agarose resin). Elution of His-Tag protein was performed with elution buffer (Imidazole, 0.5 M in 0.33x PBS). For binding to the metal Ni 2+ resin, imidazole competes with His-tag. The purification of His-tagged VEGF-C was accomplished by heparin affinity chromatography using 1M NaCl. The final step of purification was size exclusion chromatography, which resulted in size exclusion of both forms of pro-VEGF-C. The recombinant protein was diluted in PBS, filtered through a NalgeneTM syringe filter unit (Thermo Fisher Scientific, USA) and stored in 0.5x PBS at -80°C until use.
5.18.2 Thermolysin activation assay
Thermolysin protease was used to cleave pro-VEGF-C into its mature form. Different dilutions of thermolysin were used (no dilution, 1:10, 1:100, 1:1000, 1:10 000). The enzyme was reconstituted in 0.5x PBS together with purified pro-VEGF-C (0.04 mg/ml). The
72 samples were incubated for 2 days at 37 °C under aseptic conditions. After incubation, the biological activity of processed pro-VEGF-C was assessed by Ba/F3 viability assay.
5.18.3 Ba/F3 viability assay
Ba/F3 cells (a murine IL-3-dependent pro-B cell line) stably expressing VEGFR-3 were grown in DMEM/F-12 supplemented with 10 % MSC-qualified FBS, Pen/strep, and with IL-3 (0.1 ul/ml) and VEGFR-3 (0.1 ul/ml). The cells were washed three times with diluted PBS and seeded to the density of 15-10.103 cells/well in 96 well plate. Each condition was settled up intriplicates. Then cells were exposed to 200 ng/ml, 100ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.12 ng/ml of pro-VEGF-C processed by thermolysin in DMEM/F12 supplemented with Pen/Strep, 10 % mesenchymal stem cell-FBS. Cells were incubated for 48 h and thereafter 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution (0.25 mg/ml) was added to each well for 2 h incubation. After incubation, a lysis solution (stop solution) (10% SDS, 10 mM HCL) was added for 5-10 minutes, and absorbance at 540 nm was measured to reflect cell metabolic activity.