Small Scale mRNA Purification
mRNA was purified from total RNA extraction samples by oligo (dT) enrichment, using the Dynabeads mRNA purification kit (Invitrogen). Approx. 30 ug of Total RNA was adjusted to a volume of 100 ul with nuclease free water. Samples were heated to 65C for 2 minutes to disrupt secondary structure, before placing on ice. Magnetic beads were equilibrated in 100 ul of binding buffer (10 mM Tris-HCl, 1M LiCl), before addition of the 100 ul total RNA samples. Beads were incubated with RNA samples for 5 minutes at room temperature with constant rotation, before removal of the supernatant. Beads were washed twice with 200 ul of washing buffer (10 mM Tris-HCl, 1M LiCl). mRNA was eluted from the magnetic beads by addition of 10 mM Tris-HCl, pH 7.5, and its concentration determined by Nanodrop spectrophotometer.