E-MTAB-715:assay 1
Trombocyte_101213
Trombocyte_101213
RNA Extract 101213
The local Regional Ethical Review Board (Link_ping, Sweden) granted an ethical permission for this study. Unstimulated non-irradiated apheresis platelets were collected from a healthy human male blood donor. Platelets were collected by COBE Spectra system (Gambro BCT) and were used on the same collection day. Residual leukocytes were depleted with anti-CD45 conjugated Dynabeads, according to the manufacturers recommendations (Pan Leukocyte; Invitrogen, Carlsbad, CA). The platelet suspension, with a volume of 30 mL and a platelet count of 1.4 * 10exp9 cells/mL, was centrifuged at 800 g for 8 min and the supernatant was discarded. The platelet pellet was processed for leukocyte depletion, as recommended by the supplier of Pan Leukocyte reagent (Invitrogen, Carlsbad, CA). The leukocyte removal was performed at room temperature. Approximately 70 _ 75% of the original platelets were recovered after the leukocyte depletion. To investigate the level of leukocyte contamination, we determined the level of CD45 (PTPRC) transcript by qPCR using TaqMan Gene Expression Assay for this gene according to the recommendations of the supplier (Assay ID: Hs00894713_m1; Applied Bioystems, Carlsbad, CA, USA). Extraction of mRNA and synthesis of cDNA were performed by the method described by Rox JM, Bugert P, Muller J, Schorr A, Hanfland P, et al. (2004) [Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique. Clin Chem 50: 2271-2278] with the exception that we used Smarter PCR cDNA Synthesis kit for the cDNA synthesis (Clontech, Mountain View, CA, USA). Briefly, the leukocyte-depleted platelet suspension was centrifuged at 1000g for 10 min and the supernatant was discarded. Poly-A mRNA was isolated from the platelet cell pellet by using Dynabeads Oligo(dT)25 according to the instruction of the manufacturer (Invitrogen, Carlsbad, CA, USA). To synthesize the first-strand cDNA, 3.5 uL of poly-A mRNA was combined with 1uL of 3I-Smart CDS primer II A (12 uM). After mixing the tube, the sample was incubated at 72C for 3 min and then at 42C for 2 min. This was followed by the addition of a master mix containing 2 uL of 5_ First-Strand Buffer, 0.25 uL DTT (100 mM), 1 uL dNTP mix (10 mM), 1 uL SMARTer IIA oligonucleotide (12 uM), 0.25 uL RNase inhibitor, and 1 uL of SMARTScribe Reverse Transcriptase (100 U). The reverse transcription was run by incubating the tube at 42C for 1 h before the reaction was terminated at 70C for 10 min. The sample was then diluted with 90 uL of TE-buffer (10 nM Tris, 1 nM EDTA, pH 8.0). To run Long-Distance PCR, 10 uL of the diluted and reverse-transcribed platelet cDNA was mixed with 10 uL 10_ Advantage 2 PCR buffer, 2 uL 50_ dNTP mix (10 mM), 2 uL 5IPCR primer IIA (12 uM), 2 uL 50_ Advantage 2 polymerase mix, and 74 uL of deionised water to a final volume of 100 uL. The sample was then incubated in a thermal cycler running a PCR program containing 95C for 1 min, and then 20 cycles of 95C for 15 s, 65C for 30 s, and 68C for 3 min. The synthesized platelet cDNA was purified with QIAquick PCR purification kit according to the manufacturerIs instructions (Qiagen, Hilden, Germany), and the amount of cDNA was estimated on a Nanodrop spectrophotometer (ND1000; Saveen & Werner, Limhamn, Sweden).
MWG Operon, Germany
E-MTAB-715:assay 1
Experimental Factor: CELLTYPE
platelet