Drosophila S2 cells RNA extraction

Kit Used: RNeasy Mini Kit cat. no.74104 (Qiagen)

Materials:

• 70% ethanol
• 100% ethanol
• β-mercaptoethanol (β-ME)
• 1X PBS
• DEPC water
• The RNase-Free DNase Set cat. no. 79254 (Qiagen)
• QIAshredder spin-column cat. no. 79656 (Qiagen)

Procedure before starts:

• Add β-mercaptoethanol (β-ME) to Buffer RLT before use. Add 10 µl β-ME per 1 ml Buffer RLT. Buffer RLT containing β-ME can be stored at room temperature for up to 1 month. 
• Add four volumes of ethanol (100%) to the buffer RPE indicated on the bottle to obtain a working solution.
• Prepare DNase I stock solution: Resuspend the lyophilized DNase I in 550 µl of the RNase-free water or DEPC water. For long-term storage of DNase I can be aliquots and store at −20°C for up to 9 months.

Protocol Steps:

1. Pellet S2 cells (2X10⁶) by centrifugation in a centrifuge tube approximately 300 x g, for 5 min at RT.
2. Discard the supernatant. Resuspend the pellet in 1 ml 1X PBS to wash and pellet again by centrifugation at 300 x g, for 5 min.
3. Remove the supernatant and immediately add 350 µl of buffer RLT to the pellet. Lyse the cells pellet by repetitive pipetting several times. 
4. Pipet the entire lysate directly into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for 2 min at 13,000 rpm at RT.
5. Collect the lysate and add one volume of 70% (350 ul) ethanol to the lysate and mix well by pipetting.
6. Transfer up to 700 µl of the sample to an RNeasy spin column placed in a 2 ml collection tube and centrifuge for 15 seconds at 10,000 rpm at RT.
7.  Discard the flow-through. If the sample volume exceeds 700 µl, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation.
8. Add 350 µl Buffer RW1 to the RNeasy spin column, and centrifuge for 15 s at 10,000 rpm at RT. Discard the flow-through.
9. Add 10 µl DNase I stock solution to 70 µl Buffer RDD (supplied with DNase I kit) and mix by gently inverting the tube. Add the diluted DNase I (80 µl) directly to the RNeasy spin column membrane, and incubate for 15 min at RT. 
10. After the incubation, add 350 µl Buffer RW1 to the RNeasy spin column and centrifuge for 15 s at 10,000 rpm at RT. Discard the flow-through. 
11. Add 500 µl Buffer RPE to the RNeasy spin column and centrifuge for 15 s at 10,000 rpm at RT. Discard the flow-through. 
12. Wash again with 500 µl Buffer RPE to the RNeasy spin column and centrifuge for 2 min at 10,000 rpm at RT. 
13. Place the RNeasy spin column in a new 2 ml collection tube and centrifuge at 13,000 rpm for 1 min at RT to remove residual wash buffer.
14. Place the RNeasy spin column in a new 1.5 ml tube and add 30 µl DEPC water directly to the spin column membrane. Centrifuge for 1 min at 10,000 rpm at RT to elute the RNA.