P-FPMI-50 - P-FPMI-50
If isolation total RNA from cells:
1.Aspirate off supernatant and gently wash cells 2X with PBSA (22•C). Add 1 ml Trizol/ 5*106ml cells and incubate for 3 minutes.
2.Scrape cells into 1.5mL microcentrifuge tubes. Add 0.2 ul chloroform/1ml Trizol. Incubate for 5 minutes at room temperature.
3.Centrifuge at 12000 X g for 10 min at 4•C. Collect the aqueous phase, being careful not to disrupt the interface. Transfer to a fresh microcentrifuge tube and add 0.5ml isopropanol/1ml Trizol.
4.Apply 700 ul of this solution to RNeasy MINI column (Qiagen cat#74104) instructions for RNA isolation. Centrifuge at 8000X for 15 sec. Discard flow-through.
5.Wash column with 350 ul RWI buffer. Add 70 ul RNase-Free DNase Set (Qiagen cat#79254) (10 ul Rnase-free DNase and 70 ul RDD buffer) directly on to the column. Incubate for at least 15 minutes at room temperature.
6.Wash column with 350 ul RWI buffer. Centrifuge at 8000X for 15 sec. Discard flow-through.
7.Wash column with 500 ul RPE buffer. Centrifuge at 8000X for 15 sec. Discard flow-through.
8.Centrifuge at 8000X for 1 minute. Replace receptor with 1m5ml collection tube.
9.Add 50 ul RNase-free water (Sigma-Aldrich). Incubate for 1 minute, Centrifuge at 8000X for 1minute. Repeat 1.
10.Determine RNA concentration using OD260 and OD280 ratio on spectrophotometer.
11.RNA quality is assessed using Agilent 2100 Bioanalyzer and RNA 6000 Nano kits (Agilent, USA).
12.Store total RNA at -70•C.
Amplification, Extracted product
|All experiments using protocol P-FPMI-50: (E-FPMI-2, E-FPMI-3)|