This post contains a slightly modified RNeasy Kit protocol. I want to use the Qiagen RNeasy Micro Plus Kit on 4 crab hemolymph pellet samples and the four corresponding supernatant samples (GitHub Issue 577). The goal is to see if there is any RNA in the supernatant samples and if that can help increase the RNA yields for each crab sample. The supernatant samples (~1ml each) will be transfered to 15ml falcon tubes and the volumes of reagents likely need to be changed (GitHub Issue ). I will be using RNA carrier for both supernatant and pellet samples. (Sidenote: just realized I didn’t use QIAshredder columns when I did the RNeasy protocol previously. I have added that to my figure.)

Qiagen RNeasy Plus Micro Kit https://github.com/RobertsLab/resources/blob/master/protocols/Commercial_Protocols/Qiagen_RNeasy-Plus-Micro-Handbook.pdf

Prepare solutions:

Buffer RLT plus + B-ME (needs to be ratio of 10ul B-ME per 1ml Buffer RLT Plus):

40 ul of B-ME
4 ml of Buffer RLT Plus

RNA Carrier solution:

Before first-time use-
add 1ml of RNase-free H20 to dissolve the carrier RNA.
Store this stock solution in -15 to -30C.
Use stock to make fresh dilutions for each set of RNA preps

Make working solution
Add 5ul of stock solution to 34 ul of Buffer RLT plus
mix by pipetting

Add 6 ul of this diluted solution to 54ul of Buffer RLT plus to get a working solution of 4ng/ul

Protocol:

Move the ~1ml of supernatant to a 15ml falcon tube

  1. Add 350ul of Buffer RLT Plus and B-ME solution. Vortex to mix and run through a QIA shredder column 700ul at a time to further homogenize.

  2. Add 5ul of diluted carrier RNA (20 ng carrier RNA) solution and vortex 1 min.

  3. Transfer the homogenized lysate to a gDNA Eliminator spin column placed in a 2ml collection tube. Centrifuge for 30s and full speed. Discard column and save flow-through.

  4. Add 350ul of 70% ethanol to flow-through from step 3. Mix well by pipetting. Do not centrifuge.

  5. Transfer sample, including any precipitate that may have formed, to an RNeasy MinElute spin column placed in a 2ml collection tube. Close the lid gently and centrifuge for 15s at full speed. Discard flow-through.

  6. Add 700ul of Buffer RW1 to RNeasy MinElute spin column. close lid gently and centrifuge for 15s at full speed. Discard flow-through.

  7. Add 500ul of Buffer RPE to RNeasy MinElute spin column. close lid gently and centrifuge for 15s at full speed. Discard flow-through.

  8. Add 500ul of 80% ethanol to RNeasy MinElute spin column. Close lid gently and centrifuge for 2 min at full speed. Discard collection tube and flow-through.

  9. Place RNeasy MinElute spin column in new 2ml collection tube. Cut off and save lid of spin column. Place tubes open in the centrifuge and spin at full speed for 5 min. Discard collection tube and flow-through

  10. Place RNeasy MinElute spin column in new 1.5 ml collection tube. Add 14ul RNase-free water directly to center of spin column membrane. Close lid gently and centrifuge for 1 min at full speed to elute RNA.