RNA Extraction from Tissue or Cells(生物信息站-生物医学实验方法学）

RNA Extraction from Tissue or Cells

For 10 cm dish, empty the media, add 3.5 ml TRIzol Reagent, immediately scrape off the cells and pass the cell lysate many times through a 1ml papette until the lysate is not viscous.

Transfer the lysate into two 2 ml Eppendorf tubes, incubate 5 min at RT.

Add 0.4 ml chroloform into each tube, shake tubes vigorously by hand for 15 seconds and incubate them at RT for 2-3 min.

Centrifuge them at 10000-12000g for 15 min at 4°C.

Transfer the aqueous phase (supernatant), add 0.8-1 ml isopropanol and incubate at RT for 10 min, centrifuge at 10000-12000g for 10 min at 4°C. Remove the supernatant and keep the pellet.

Wash the RNA pellet with 2 ml 75% alcohol twice by centrifugation at 7500g for 5 min at 4°C.

Briefly air dry the RNA pellet (don’t dry the RNA completely), dissolve it in RNase-free H2O, add 2 ul RNase inhibitor.

Optional 1: Add 0.5 ml TRIzol reagent, re-extract the RNA to reduce the DNA contamination.
Optioanl 2: Add 1-2 ul RNase-free DNase I and 3 ul RNase inhibitor, incubate it at RT for 30 min, purify it using RNeasy Mini Kit (100 ug capacity), QIAGEN, Cat# 74104.
----Dilute the sample to 100 ul by adding RNase-free H2O
----Add 350 ul Buffer RLT, mix (no Vortex)
----Add 250 ul 100% ethanol, mix gently
----Apply the sample (700ul) to a RNEasy Column in a 2ml collection tube
----Centrifuge 15 sec at 10000g, discard the flowthrough and collection tube
----Transfer the column to a new 2 ml tube
----Add 500 ul Buffer RPE to wash 15 sec at 10000g
----Add another 500 RPE to wash 2 min at 10000g to dry
----Transfer the column to a new tube, centrifuge 1 min at full speed to further dry
----Transfer the column to a new 1.5 ml tube, pipette 30 ul-50ul RNase-free H2O, wait for 1-2 min, centrifuge for 1 min at 10000g to elute RNA. Also, elute it use another 20 ul water.
8. Briefly air dry the RNA pellet (don’t dry the RNA completely), dissolve it in RNase-free H2O, add 3 ul RNase inhibitor.