This protocol describes how to clean/concentrate RNA with the Zymo RNA Clean & Concentrator-5 kit.

• Astrangia 2021 master extraction sheet
• Clean + Concentrate test sheet (JA & ZD samples)

Materials & Equipment

• Zymo RNA Clean & Concentrator -5 kit
  • From kit:
    • RNA binding buffer
    • RNA prep buffer
    • RNA wash buffer
    • DNase/RNase free water
    • Spin IC columns and collection tubes
• Centrifuge and rotor capable of spinning at 15,000 rcf
• Plastics
  • 2 1.5 mL microcentrifuge tubes per sample
  • 1 quibit tube per sample
  • Several 1000 µL, 200 µL, and 20 µL filtered pipette tips per sample

Samples

Today, I tested out the RNA clean/concentrate kit on 4 of my samples with low RNA concentrations. I need 50 ng/µL to send to Azenta for RNA sequencing (small, long, mRNA).

These were the samples I used:

Sample	Avg RNA ng/µL	µg	Extraction date
AST-2523	20.1	1.809	11/7/22
AST-1065	16.3	1.467	11/7/22
AST-1761	45.8	4.122	1/12/23
AST-2403	30.6	2.754	1/16/23

Each sample had ~90 µL of RNA in the tube.

Protocol

• Add 2 volumes of RNA binding buffer to each sample and pipette up and down to mix
  • 40 µL of sample; 80 µL of buffer
• Add equal volume of 100% ethanol and pipette up and down to mix
  • 120 µL of ethanol
• Transfer sample to spin column and spin at 15,000g for 60 seconds. Discard flow-through
• Add 400 µL of RNA prep bugger and spin at 15,000g for 60 seconds. Discard flow-through
• Add 700 µL of RNA wash bugger and spin at 15,000g for 60 seconds. Discard flow-through
• Add 400 µL of RNA wash bugger and spin at 15,000g for 60 seconds. Discard flow-through
• Move spin column to 1.5 mL tube
• Add 15 µL of DNase/RNase free water to column and spin at 15,000g for 60 seconds - this is the cleaned & concentrated RNA!

Quality control

Quibit results

Tube	Type	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Avg (ng/uL)	PRIOR Avg (ng/uL)
S1	Standard	409.79	NA
S2	Standard	8736.12	NA
AST-2523	Sample	32	31.8	31.9	20.1
AST-1065	Sample	28.4	28	28.2	16.3
AST-1761	Sample	65.4	65.8	65.6	45.8
AST-2403	Sample	44.2	44	44.1	30.6

Here’s a table that Zoe & I put together that outlines our expected v. observed results.

A) SampleID	B) AvgConcentration (ng/uL)	C) ng	D) Take 40 uL from each	E) Expected concentration in 15 uL	F) Avg concentration post-kit	G) ng post-kit	% RNA recovered
AST-2523	20.1	2010	804	53.6	31.9	478.5	0.5951493
AST-1065	16.3	1630	652	43.4666667	28.2	423	0.648773
AST-1761	45.8	4580	1832	122.133333	65.6	984	0.5371179
AST-2403	30.6	3060	1224	81.6	44.1	661.5	0.5404412
Calculations		=B*100	=C*0.4	=D/15		=H*15	=G/D

Things to test:

• Letting DNase/RNase free water sit on spin column for 1-2 mins
• Eluting in 25 µL instead of 15 µL