Microarray hybridization and you will checking, studies buy and investigation

Microarray hybridization and you will checking, studies buy and investigation

Total RNA is removed with the TRIZOL reagent according to the maker’s method. RNA is actually quantified playing with a good spectrophotometer and its own top quality is actually featured by the agarose gel electrophoresis and also by the new Agilent 2100 Bioanalyzer platform, pursuing the manifacturer’s recommendations to have take to planning and you will studies of information (Agilent 2100 Bioanalyzer 2100 Specialist Customer’s Publication).

Polysomal RNA extraction

Cells were washed once with phosphate buffer saline (PBS + cycloheximide 10 ?g ml -1 ) and treated directly on the plate with 300 ?l lysis buffer [10 mM NaCl, 10 mM MgCl2, 10 mM Tris–HCl, pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.2 U ?l -1 RNase inhibitor (Fermentas), cycloheximide 10 ?g ml -1 and 1 mM dithiothreitol] and transferred to an Eppendorf tube. After a few minute incubation on ice with occasional vortexing, the extracts were centrifuged for 5 min at 12,000 g at 4 °C. The supernatant was stored at ?80 °C or loaded directly onto a 15–50% linear sucrose gradient containing 30 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and centrifuged in an Sorvall rotor for 100 min at 180,000 g.

Read moreMicroarray hybridization and you will checking, studies buy and investigation