Concentrating RNA samples using Vacufuge

For the RISG project, we decided to pool the three replicate RNA samples for each treatment before moving forward with mRNA Sequencing Library Prep. For mRNA Seq Prep, the desired starting input was 1200 ng of RNA concentrated in 25 μL.

For 10 pooled sample sets, the pooled volume to achieve 1200 ng of RNA was greater than 25 μL. A vacufuge was used to concentrate these samples down into 25 μL.

Vacufuge sample preparation

• Before pooling samples, make new 1.5 mL tubes
  • I added 25 μL of nuclease-free water into each tube, marked the outside of the tube at the water line with a Sharpie, and then pipetted out the water
  • This was used as a visual guide when vacufuging the samples
• For each of the 3 RNA samples going into the pool, aliquot 500 ng of RNA
  • I decided to vacufuge 1500 ng of RNA, in case there was any loss of RNA during the vacufuge process
• Place tubes in vacfuge and use the following settings:
  • Brake: ON
  • Temp: Ambient (–)
  • Mode: V-AQ
  • Time: (this will depend on the starting volume in the samples)
    • I started with 15 minutes and recorded the final time it took to vacufuge samples to 25 μL

Sample	Aliqout for 1500 ng (μL)	Vacufuge time (minutes)
B2_TSE1_Con	60.37	40
B2_TSE1_ConSE	48.30	30
B2_TSE1_Hi	80.28	50
B2_TSE1_HiSE	64.70	40
B3_TSE1_Hi	35.07	15
B3_TSE1_HiSE	34.13	15
B4_TCE_HB	44.98	30
B4_TSE1_Con	36.59	15
B4_TSE1_Hi	31.69	15
B4_TSE1_HiSE	39.98	30

RNA BR Assay Qubit

Sample	Qubit 1 (ng/μL)	Qubit 2 (ng/μL)
Std. 1	76.39
Std. 2	5547.23
B2_TSE1_Con	59.8	60.2
B2_TSE1_ConSE	61.2	61.2
B2_TSE1_Hi	58.0	58.0
B2_TSE1_HiSE	55.4	56.0
B3_TSE1_Hi	59.4	60.2
B3_TSE1_HiSE	61.6	62.2
B4_TCE_HB	83.2	83.0
B4_TSE1_Con	58.8	56.8
B4_TSE1_Hi	67.4	67.4
B4_TSE1_HiSE	88.8	89.2

I seemed to have over-vacufuged 4 of the pooled samples, I don’t have enough volume to achieve 1200 ng.

• Samples:
  • B2_TSE1_Con
  • B2_TSE1_Hi
  • B2_TSE1_HiSE
  • B4_TSE1_HiSE

I re-did these four samples:

Sample	Vacufuge time (minutes)
B2_TSE1_Con	30
B2_TSE1_Hi	36
B2_TSE1_HiSE	33
B4_TSE1_HiSE	20

RNA BR Assay Qubit

Sample	Qubit 1 (ng/μL)	Qubit 2 (ng/μL)
Std. 1	75.49
Std. 2	5606.77
B2_TSE1_Con	55.4	55.8
B2_TSE1_Hi	45.0	45.0
B2_TSE1_HiSE	65.8	65.8
B4_TSE1_HiSE	72.6	73.0

Again, I did not have enough volume to achieve 1200 ng. I decided to take 600 ng from each vacufuge round and combine them.

Sample	Aliquot for 600 ng (Vacufuge round 1)	Aliquot for 600 ng (Vacufuge round 1)	Final Volume
B2_TSE1_Con	10.03 μL	10.83 μL	20.86 μL
B2_TSE1_Hi	10.34 μL	13.33 μL	23.68 μL
B2_TSE1_HiSE	10.83 μL	9.12 μL	19.95 μL
B4_TSE1_HiSE	6.76 μL	8.26	15.02 μL

These 10 samples are ready to go for mRNA Seq Library Prep.