Mrna isolation kit for blood bone marrow

The blood samples used for pharmacodynamic monitoring were obtained in parallel with the blood samples for pharmacokinetic monitoring and were analyzed at Vall d’Hebron Research Institute.
To do so, blood samples were collected in BD Vacutainer^® lithium heparin tubes. One milliliter of heparinized blood was stimulated with 1 mL of complete RPMI 1640 (Gibco) enriched with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) and 5 µg/mL of ionomycin (Sigma Aldrich, Saint Louis, MO) for three hours at 37°C and 5% CO₂. Then, 10 mL of RNA/DNA Stabilization Reagent for Blood/Bone Marrow (Roche, Mannheim, Germany) were added to lyse cells and stabilize nucleic acids, according to the manufacturer’s instructions. Samples were stored at -20°C for a maximum of a year.
mRNA was isolated using an mRNA Isolation Kit for Blood/Bone Marrow (Roche, Mannheim, Germany) following the manufacturer’s instructions. It was then stored at -20°C for a week or less, or at -80°C indefinitely.
Reverse transcription was performed with 8.2 µL of mRNA in a thermocycler using avian myeloblastosis virus reverse transcriptase and oligo (dT) as a primer (First Strand cDNA synthesis kit Roche, Mannheim, Germany) following the manufacturer’s instructions. The resulting cDNA was stored concentrated at -20°C until further use.