Total RNA Purification by Quick-RNA MiniPrep (Zymo Research)

Dissect flies in PBS, or collect undissected flies into 1.5 ml microcentrufuge tube.

Prior to use, add ethanol to the RNA Wash Buffer concentrate (R1003-3-48). See bottle instructions
Perform all steps at room temperature and centrifuge at 10,000 - 16,000 x g. 

Homogenize in 300 ul RNA Lysis Buffer (R1060-1-100). 

Clear lysate by centrifugation at ≥10,000 x g for 1 min. 

Transfer the supernatant into a Spin-Away Filter (C1006-50-F) in a Collection Tube (C1001-50) and centrifuge at ≥10,000 x g for 1 min to remove the majority of gDNA. 

Add 1 volume ethanol (95-100%) to the flow-through and mix well. 

Transfer the mixture to a Zymo-Spin IIICG Column (C1006-50-G) and centrifuge 30-45 sec. 

Recommended Step: DNase I treatment (in column)

Add 400 ul RNA Prep Buffer to the column and centrifuge for 45 sec. Discard the flow-through.

Add 700 ul RNA Wash Buffer to the column and centrifuge for 45 sec. Discard the flow-through.

Add 400 ul RNA Wash Buffer and centrifuge the column for 2 min to ensure complete removal of the wash buffer.

Transfer the column carefully into an RNase-free tube add DNase/RNase-Free Water directly into the column matrix and centrifuge for 45 sec.

Eluted RNA can be used immediately or stored at -80°C.

Measure the RNA concentration by NanoDrop.