2023-05-05 Pooling RNA Using Zymo Clean and Concentrate Kit for ENCORE Project

Concentrating Low-Quantity ENCORE RNA and Pooling Re-extractions Using Zymo Clean and Concentrate Kit for ENCORE Project, May 5, 2023

After finishing re-extractions for the super-low-quantity samples for the ENCORE project on 4/27/23, I used the Zymo RNA Clean and Concentrate Kit as also used previously for this project.

Today I did two things:

  1. Concentrated the RNA that had a concentration between the range of 7-11 ng/uL to get a concentration of at least 12.

  2. Pooling re-extractions with initial extractions when the re-extractions on 4/27/23 still had a concentration less than 12 ng/uL.

Protocol

For both of these conditions, the protocol was the same but for the pooling I just took the entire volume of the initial extraction and combined it with the entire volume of the re-extraction, then performed the Zymo protocol using the total combined volume.

Then, follow the protocol based on the volume for each tube for the Zymo Clean and Concentrate kit, eluting in 25 µL of DNAse/RNAse free water.

For an example tube where two RNA tubes of 40 uL (initial extraction and re-extraction) are combined to make 80uL (or just an RNA tube with a volume of 80uL):

Base volume: 80 uL

  1. Since each sample is 80 uL, add 160 uL (2 x 80) of RNA binding buffer.
  2. Add an equal volume (240 uL (3 x 80)) of 100% ethanol and mix.
  3. Transfer the sample to the Zymo-Spin™ IC Column in a Collection Tube and centrifuge. Discard the flow-through.
  4. Add 400 µl RNA Prep Buffer to the column and centrifuge. Discard the flow-through.
  5. Add 700 µl RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
  6. Add 400 µl RNA Wash Buffer to the column and centrifuge for 1 minute ensure complete removal of the wash buffer. Carefully, transfer the column into a RNase-free tube (not provided).
  7. Add 25 µl DNase/RNase-Free Water directly to the column matrix and centrifuge.

Example for a base volume of 90 uL:

Base volume: 90 uL

  1. Since each sample is 90 uL, add 180 uL (2 x 90) of RNA binding buffer.
  2. Add an equal volume (270 uL (3 x 90)) of 100% ethanol and mix.
  3. Transfer the sample to the Zymo-Spin™ IC Column in a Collection Tube and centrifuge. Discard the flow-through.
  4. Add 400 µl RNA Prep Buffer to the column and centrifuge. Discard the flow-through.
  5. Add 700 µl RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
  6. Add 400 µl RNA Wash Buffer to the column and centrifuge for 1 minute ensure complete removal of the wash buffer. Carefully, transfer the column into a RNase-free tube (not provided).
  7. Add 25 µl DNase/RNase-Free Water directly to the column matrix and centrifuge.

Samples

1. Concentrating just original extraction

Extraction.Date Extraction.Notes Volume in Tube currently colony_id
20230118   90 Dlab-A3
20221017   90 Dlab-A5
20230216   90 Dlab-B1
20230113 Sent 40 uL in pool for Transcriptome 50 Dlab-B5
20221005 Sent 40 uL in pool for Transcriptome 50 Dlab-C7
20230421   90 Mcav-A5
20230425   90 Mcav-B5
20230418   90 Mcav-C5
20230118   90 Mcav-D6
20230118   90 Mcav-E5
20221005   90 Mcav-E6
20230425   90 Mcav-F5
20221005   90 Mcav-F7
20230425   90 Mdec-A1
20221017   90 Mdec-A4
20230113   90 Mdec-D6

2. Pooling initial and re-extraction

Extraction.Date Extraction.Notes Volume in Tube currently colony_id
20230418   40 Dlab-A7
20230427   40 Dlab-A7
20230421   40 Dlab-D6
20230427   40 Dlab-D6
20230421   40 Dlab-E4
20230427   40 Dlab-E4
20230406 Very little tissue in tube 35 Dlab-E6
20230427 Very little tissue in tube 40 Dlab-E6
20230216   90 Dlab-F3
20230427   40 Dlab-F3
20221017   90 Dlab-F4
20230427   40 Dlab-F4
20230113   90 Dlab-F5
20230427   40 Dlab-F5
20221003   90 Dlab-F6
20230427   40 Dlab-F6
20221017   90 Mcav-B6
20230427   40 Mcav-B6

Qubit Results

  • Used High Sensitivity range dsDNA and RNA Qubit Protocol
  • All samples read twice, standard only read once

RNA Standards: 33.21 (S1) & 438.03 (S2)

1. Concentrating just original extraction

colony_id RNA_QBIT_AVG Volume in Tube currently Amount RNA in whole tube
Dlab-A3 13.55 24 325.2
Dlab-A5 15.65 24 375.6
Dlab-B1 12.35 24 296.4
Dlab-B5 16.05 24 385.2
Dlab-C7 17.15 24 411.6
Mcav-A5 17.1 24 410.4
Mcav-B5 18.1 24 434.4
Mcav-C5 14.8 23 340.4
Mcav-D6 19 24 456
Mcav-E5 17.45 24 418.8
Mcav-E6 17.25 24 414
Mcav-F5 18.55 24 445.2
Mcav-F7 15.3 24 367.2
Mdec-A1 13.3 24 319.2
Mdec-A4 15.85 24 380.4
Mdec-D6 15.8 24 379.2

2. Pooling initial and re-extraction

colony_id RNA_QBIT_AVG Volume in Tube currently Amount RNA in whole tube
Dlab-A7 17.55 24 421.2
Dlab-D6 13.4 24 321.6
Dlab-E4 14.1 24 338.4
Dlab-E6 12.25 23 281.75
Dlab-F3 19.1 24 458.4
Dlab-F4 18.9 24 453.6
Dlab-F5 14 24 336
Dlab-F6 25.5 24 612
Mcav-B6 17 24 408

All are now above 12 ng/uL and ready for plate prep!

Written on May 5, 2023