2.1 Blood sampling and RNA isolation from peripheral blood mononuclear cells (Studies I-IV)
PBMC were separated in a VACUTAINER CPT cell preparation tube (Becton Dickinson and Company, Franklin Lakes, N.J., USA) according to the manufacturer’s protocol. Total cellular RNA was isolated from stored cell lysate with Qiagen’s RNase minikit (QIAGEN GmbH, Hilden, Germany) according to the
manufacturer’s instructions. DNA was removed with Qiagen’s RNase-Free DNase set. Total RNA was eluted to nuclease-free water and samples were stored -70 ºC.
Sera were separated from blood and stored at -70 ºC.
2.2 Gene expression analysis by Lightcycler (Study I)
The expression of TRAIL mRNA could be determined in 46 patients and 17 controls in whom RNA was available. A relative quantitative real time polymerase chain reaction (RT-PCR) was performed with a LightCycler instrument (Roche Diagnostics GmbH, Mannheim, Germany). 1 µg of total RNA was reverse transcribed to complementary DNA (cDNA) with a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) following standard protocol with random hexamer primers.
Table 7. TRAIL primers and probes
Primer/Probe Sequence
Forward primer 5’-AGAGAGTATGAACAGCCCCTG-3’
Reverse primer 5’-GTCCCAGTTATGTGAGCTGCTA-3’
Probe 1 5’-Red640-TTCTAACGAGCTGACGGAGTTGCCA-3’
Probe 2 5’- GGTTTCCTCAAGAGGTTCTCAAAAATCATC-FL-3’
Abbreviations: A adenine; C cytosine; G guanine; T thymine.
2-µl cDNA was used in a total of 20 µl reaction volume for the PCR reactions.
The final concentrations of the target reagents in the system were: 1 x LightCycler FastStart DNA Master HybProbe, 4 mM MgCl2, 0,5 µM each primer and 0,2 µM each probe. PCR cycling conditions were: denaturation at 95 ºC for 10 min, amplification 45 cycles, at 95º C for 15 seconds, at 55 ºC for 15 seconds and at 72 ºC for 15 seconds and cooling at 40 ºC for 30 seconds. Primers and probes for the LightCycler PCR were designed and prepared by TIB MolBiol (Berlin, Germany) (Table 7). As a reference gene we used human glucose-6-phosphate-dehydrogenase (h-G6PDH). Reference gene PCR was done at the same time and the same PCR conditions as the target gene by a LightCycler h-G6PDH housekeeping gene set kit (Roche Diagnostic GmbH, Mannheim, Germany). The reference gene was selected by a LightCycler h-Housekeeping Gene Selection Set, which included five alternative reference genes. All reactions for target and reference genes were made as
duplicate. Quantitative RT-PCR results were calculated by a LightCycler Relative Quantification Software with efficiency correction (Roche Diagnostics GmbH).
2.3 QRT-PCR using low density array (Study III)
Total-RNA (1 µg) was reverse transcribed to cDNA using High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) with standard protocol. Gene expression analyses were done with TaqMan low-density array (LDA) (Applied Biosystems) using 7900HT Real-time PCR system (Applied Biosystems). Human apoptosis array was used to determine expression of 93 apoptotic-molecules and three housekeeping genes from PBMC. Arrays were loaded with 4 l undiluted cDNA, 42 l H2O, and 50 l PCR Universal master mix and run according to the manufacturer’s instructions. Samples were run as duplicate.
The expression data was analyzed with RQ manager software using comparative Ct method ( Ct). To normalize the results, the housekeeping gene glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was used and two healthy control samples were used as calibrator in the data analysis.
2.4 Flow cytometry analysis (Study I)
PBMC for TRAIL membrane expression analyses was obtained from 9 RRMS patients and 6 healthy controls. PBMCs (2 x10 5) were incubated with mouse anti- human CD3-APC-Cy7, CD4-PE-Cy7, CD8-APC, CD14-FITC and TRAIL-PE (Becton Dickinson, Franklin Lakes, NJ, USA) monoclonal antibodies for 20 min. As an isotype control, cells were incubated with mouse anti-human IgG1-APC-Cy7, IgG1-PE-Cy7, IgG1-APC, IgG1-FITC and IgG1-PE monoclonal antibodies.
Thereafter cells were washed twice, resuspended in PBS, and analyzed with FACSAria flow cytometry and Facsdiva software (Becton Dickinson). Stainings were made in duplicate. Optimization of fluorescence compensation was made using BD CompBeads (Becton Dickinson).
2.5 Enzyme-linked immunosorbent assay (Studies I, II and IV)
Levels of sTRAIL in undiluted sera were quantified by a Diaclone solid phase sandwich ELISA kit (# 850.770.096; Diaclone, Besancon Cedex, France). Detection limits were 64 pg/ml. TRAIL absorbancies were read with a Multiskan MS version 4.0 spectrophotometer (Labsystems, Helsinki, Finland) at wavelength 450 nm.
2.6 Luminex (Studies I, II and IV)
The collected blood was allowed to clot for at least 30 minutes before separation.
The blood containing tubes were then centrifuged for 15 minutes at 1600 x g. Sera were separated from the blood, aliquoted and stored at -70 C.
The levels of sFas, sFasL and MIF were measured with a Human sepsis/apoptosis LINCOplex Kit (Linco Research, St. Charles, Missouri, USA) (Studies I, II, and IV), CCL2, CCL3, CCL4 and CXCL10 with a Human cytokine LINCOplex kit (Linco Research) (Study II) and IL-2, IL-6, IL-10, IL-12p70, IFN- and TNF- with High Sensitivity Human cytokine LINCOplex kit (Linco Research) (Study II). All the data were collected and analyzed using Bio-Plex suspension array system and Bio-Plex Manager software 4.1 (Bio-Rad laboratories, California, USA). A five- parameter regression formula was used to calculate the sample concentration from the Human sepsis/apoptosis LINCOplex Kit and the High Sensitivity Human cytokine LINCOplex kit, and a four-parameter regression formula was used for the Human cytokine LINCOplex kit. All 96 well plates included samples from all disease subtypes and controls to minimize inter-assay variation. The same batch of monoclonal antibodies for the Bio-Plex Cytokine Assay System was used throughout the experiments; the inter-assay and intra-assay values are reported to be less than 15% by the manufacturer. The percent recovery of standards ranged from the 90% to 110% that was used as a detection limit for each protein. The lower detection limits were as follows: 12.2 pg/ml for sFas, sFasL and MIF, 16.0 pg/ml for CXCL10, CCL3 and CCL4, 3.2 pg/ml for CCL2, 0.13 pg/ml for IL-10, TNF-a, IL-6, IL-12p70, IL-2 and IFN-g.