P-FPMI-142 - P-FPMI-142

RNeasy Mini Kit Qiagen: Cat # 74104/74106 was used accoding to manufacturers instructions to isolate RNA from PBM. In brief, lyse PBM (on magnetic beads) in 1.2 ml lysis buffer containing beta-mercaptoethanol. Add 1.2 ml of 70% ethanol to the suspension, mix well by pipetting. Apply 600-750 ul of sample to the RNeasy mini column placed in a 2 ml collection tube. Centrifuge for 15 sec at 8000 x g and discard the flow through. Apply more sample to the mini column, centrifuge and discard flow through until all sample is loaded on the column. Pipette 350 ul buffer RW1 into the mini column and centrifuge for 15 sec at. Add 120 uls per column of DNase 1 in Buffer RDD (RNase-free DNase I (Qiagen cat # 79254) made to 1500 Kunitz units/550ul RNAse free H2O) and allow digestion to proceed for 20 mins at room temperature. Pipette 350 ul Buffer RW1 into the mini column and centrifuge for 15 sec at 8000 x g. Transfer the RNeasy column to a new 2 ml collection tube, pipette 500 ul Buffer RPE onto the column, centrifuge for 15 sec at 8000 x g. Discard flow thru and repeat wash twice. Centrifuge for 2 min at 8000 x g, place the column in a new 1.5 ml collection tube, pipette 30 ul RNase-free water directly onto the silica-gel membrane. Centrifuge for 1 min at 8000 x g. Repeat elution with an additional 30 ul of RNase-free water (total elution volume of 60ul). Add 1 ul RNase Inhibitor to your sample. Aliquot RNA and store at -80C. Analyze quality and quantity of RNA on Agilent Bioanlyzer.
Amplification, Extracted product
All experiments using protocol P-FPMI-142: (E-FPMI-7, E-FPMI-9)