Grace’s Notebook: 6 pooled Crab RNA samples – unsuccessful concentration with Zymo RNA Clean and Concentrator Kit -5

Today I tried to concentrate the 6 pooled crab RNA samples. Long story short, a lot of RNA was lost in the process and the “concetrated” samples are now well below the requirements for NWGC for RNAseq. Details in post. Next steps, I’m going to extract more RNA from samples from Day 9 so we can bulk those pools up if we want to try to send them to NWGC still. Then I’ll extract more RNA from samples that will bulk up the other pools, too.

6 pooled samples

The initial RNA concentrations for the 6 pooled samples are: (post: here)

trtmnt_tank day infection_status total-yield_ng elution_vol vol_for_pool total_vol_ul qubit_vol_ul qubit_sample_ng total_ng_RNA_pool final_sample_vol
NA 9 0 287.3 13 4.977375566 52.65550411 2 21 1063.765586 50.65550411
NA 9 1 305.5 13 4.680851064 57.13842355 2 21.5 1185.476106 55.13842355
cold 12 0 412.1 13 3.470031546 51.61757335 2 23.6 1170.974731 49.61757335
cold 12 1 280.8 13 5.092592593 39.22471819 2 27.2 1012.512335 37.22471819
warm 12 0 380.9 13 3.754266212 40.86453844 2 26.9 1045.456084 38.86453844
warm 12 1 230.1 13 6.214689266 41.00446376 2 26.3 1025.817397 39.00446376

NWGC requirements

They require normalized at least 1.5 micrograms (15000 ng) of RNA. Each sample should be concentrated to 50ng/ul of RNA. Each sample should be eluted (normalized) in at least 30ul of TE.

Attempt at concentrating

Earlier in the week I tried concentrating two test pools that I made. Post: here. I made a mistake because I diluted the test pools before running 2ul on the qubit, so I didn’t have a baseline idea of how much RNA was in the samples.

Today’s attempt

I know the conenctration and volume of all the samples (in table above).

Because there was a sample that had 55.14ul in it, I decided to normalize them all to 60ul by adding RNAse-free water.

Then I went through the protocol as described.

  1. Add 100ul of RNA Binding Buffer to each sample. Mix by pipet. (Current volume now 160ul).
  2. Add 1 volume (160ul) of 100% ethanol. Mix by pipet. Transfer to a Zymo-Spin IC Column (provided). Centrifuge at 10,000 g for 30s. Discard flow-through.
  3. Add 400ul of RNA Prep Buffer to the column. Centrifuge 10,000g for 30s. Discard flow-through.
  4. Add 700ul of RNA Wash Buffer to the column. Centrifuge 10,000g for 2 minutes. Discard flow-through. Transfer column to labeled RNAse-free microcentrifuge tube.
  5. Add 35ul of TE to the column. Centrifuge 10,000g for 2 minutes. RNA is now eluted in the TE.

Qubit results

I ran 2ul of each sample on the qubit using RNA HS Kit.
The concentrations were wayyyy too far below the required 50ng/ul. And each sample lost RNA, some more than others. 😦

Day Infection status Temperature RNA ng/ul in 2ul Total RNA in sample ng (33ul of sample remaining)
9 0 NA 15.7 518.1
9 1 NA 17.6 580.8
12 0 cold 24.9 821.7
12 1 cold 26.0 858
12 0 warm 26.0 858
12 1 warm 24.4 805.2

Next steps

On Tuesday, January 14th I’ll extract RNA from another set of 24. I’ll focus on Day 9 infected and uninfected again, so that if we want to bulk up those 6 pooled samples with more RNA to send to NWGC, we can.

from Grace’s Lab Notebook