Laura’s Notebook: Oly OA RNA isolation – Ctenidia

July 12, 2019

Homogenized the rest of the Oly ctenidia tissues, as described in the previous post. For most samples I acheived at minimum 30 mg tissue. The scale was not very sensitive, so I’m estimating an error of +/- 20mg.

Tested the RNAzol isolation process on 6 samples, one from each cohort x treatment. Followed the RNAzol® RT RNA Isolation Reagent protocol for Total RNA isolation, using half of my homogenate, so 0500 uL.

RNAzol Steps:

  • Labeled 1.7 mL tubes with sample numbers, 2 tubes per sample
  • Added 200 uL DEPC-treated water to one set of tubes
  • Transferred 500 uL of homogenate to tubes with water, returned rest of homogenate (500 uL) to -20 freezer.
  • Vortexed homogenate + water vigorously 15 seconds
  • Held mixture for 15 minutes at room temperature
  • Centrifuged at 12,000 g (aka rcf) for 15 minutes. NOTE: for this first batch of 6 samples, I accidentally centrifuged at 1,200 for 15 minutes, and didn’t catch the error until later.
  • Transfered supernatant to new tubes (used 2nd set of labeled tubes). DNA/proteins/polysaccharides remain in the bottom of the tubes. I added the label “DNA + Pr.” to these tubes, and saved in the -20 freezer.
  • Added 0.75 mL isopropanol to precipitate RNA
  • Vortexed vigorously for 15 seconds. Held for 10 minutes at room temperature
  • Centrifuged at 12,000 rcf for 10 minutes
  • Discarded supernatant. A white RNA pellet was visible in samples (which is good)
  • Added 500 uL 75% ethanol, which I prepared using the 200 proof ethanol and DEPC-treated water.
  • Mixed by hand, made sure the pellete was in the ethanol, then centrifuged at 8,000 rcf for 3 minutes.
  • Discarded ethanol supernatant, repeated previous step, then discarded ethanol again.
  • Added 50 uL DEPC-treated water, vortexted by hand for 10 seconds.
  • Stored RNA dissolved in water in the -20 freezer for now.

July 15, 2019

Another batch of RNA isolation today. I ran 24 samples at once, which I won’t do again, as it takes too long per step. Otherwise, things went well and there were no changes to the above protocol. One note: sample 295 had no visible RNA pellet, so I don’t expect I’ll find any RNA when I quantify. This was probably due to the small size of the initial tissue piece, and loss during mortar/pestle homogenization.

July 16, 2019

Finished isolating ctenidia RNA today. Ran 2 batches of 12 samples, which allowed me to finish more quickly, and ultimately didn’t take much longer tha doing 24 in one batch. Good to know for the future. Again, no changes to protocol, but some notes:

  • The last batch accidentally sat for 25 minutes during the initial homogenate + water step (should have been 15 minutes) – timer malfunction :/
  • Dissolved RNA pellet in 150 uL DEPC treated water, not 50 uL. See notes below.

RNA Quantification, round 1

Quantifed 18 samples, 3 from each treatment x cohort. Used 5 uL (of 50 uL), and the Qubit RNA assay kit (high sensitivity). All except 1 sample (#302) read “TOO HIGH”. I chatted with Sam, and based on the size of my RNA pellets he suggested dissolving RNA in 150 uL water, not 50 uL, and using 1 uL with quantifying.

Based on the above, I added 100 uL of DEPC-treated water to all samples that I had previously processed. NOTE: this means that the 18 samples that I already quantified now have a total volume of ~145 uL, while the rest will have 150 uL, with a few exceptions:

  • I did not add more water to sample 302, as it quantified in the first round.
  • Samples 295, X and Y: only added 50 uL water, as no pellets were visible.

RNA Quantification, round 2

Quantified all ctenidia samples (except for #302) using Qubit HS RNA assay. Prepared the 53 samples, plus the two standards. Thawed samples, then transferred 1 uL into Qubit working solution (prepared using 60 uL reagent + 11,940 uL buffer). Good news is that I have enough RNA in all samples – see table in repo, also pasted below – need to dilute a few samples and re-quantify before proceeding to the QuantSeq library prep.

Cohort pCO2 HOMOGENATE TUBE # TUBE COLOR TISSUE TYPE TISSUE SAMPLE # VOL RNAzol MASS TISSUE (mg) DATE HOMOGENIZED Homogenization Batch DATE RNA ISOLATED RNA Isolation Batch Total volume, uL (RNA + H2O) [RNA] ng/uL Amount of RNA (ng) Volume needed for 500 ng RNA Notes
Dabob Bay High 291 PURPLE CTENIDIA HL10-10 1 mL 80-100 7/10/19 1 7/12/19 1 144 200 28,800 2.50
Dabob Bay High 292 PURPLE CTENIDIA HL10-11 1 mL 10-30 7/10/19 1 7/15/19 2 144 47.8 6,883 10.46
Dabob Bay High 293 PURPLE CTENIDIA HL10-12 1 mL 10-30 7/10/19 1 7/15/19 2 144 47.2 6,797 10.59
Dabob Bay High 294 PURPLE CTENIDIA HL6-10 1 mL 80-100 7/10/19 4 7/15/19 2 149 108 16,092 4.63
Dabob Bay High 295 PURPLE CTENIDIA HL6-11 1 mL 10-30 7/10/19 4 7/15/19 2 49 49.6 2,430 10.08
Dabob Bay High 296 PURPLE CTENIDIA HL6-12 1 mL 60-80 7/12/19 5 7/16/19 3 149 198 29,502 2.53
Dabob Bay High 297 PURPLE CTENIDIA HL6-13 1 mL < 10 7/12/19 5 7/16/19 3 149 4.2 626 119.05
Dabob Bay High 298 PURPLE CTENIDIA HL6-14 1 mL 80-100 7/12/19 7 7/16/19 4 149 76.4 11,384 6.54 Black substance in homogenate – maybe from tube lid?
Dabob Bay High 299 PURPLE CTENIDIA HL6-15 1 mL 10-30 7/12/19 7 7/16/19 4 149 56.8 8,463 8.80
Dabob Bay Ambient 301 RED CTENIDIA HL10-19 1 mL 10-30 7/10/19 1 7/12/19 1 144 82.6 11,894 6.05
Dabob Bay Ambient 302 RED CTENIDIA HL10-20 1 mL 10-30 7/10/19 1 7/15/19 2 45 36.8 1,656 13.59
Dabob Bay Ambient 306 RED CTENIDIA HL10-21 1 mL 50-70 7/10/19 2 7/15/19 3 144 200 28,800 2.50
Dabob Bay Ambient 304 RED CTENIDIA HL6-19 1 mL 50-70 7/10/19 1 7/15/19 2 149 TOO HIGH >28,000 #VALUE!
Dabob Bay Ambient 305 RED CTENIDIA HL6-20 1 mL 20-40 7/10/19 1 7/15/19 2 149 82 12,218 6.10
Dabob Bay Ambient 303 RED CTENIDIA HL6-21 1 mL 80-100 7/12/19 1 7/16/19 2 149 120 17,880 4.17
Dabob Bay Ambient 307 RED CTENIDIA HL6-16 1 mL 70-100 7/12/19 4 7/16/19 3 149 95.4 14,215 5.24
Dabob Bay Ambient 308 RED CTENIDIA HL6-17 1 mL 70-90 7/12/19 4 7/16/19 4 149 63.6 9,476 7.86
Dabob Bay Ambient 309 RED CTENIDIA HL6-18 1 mL 80-100 7/12/19 6 7/16/19 4 149 200 29,800 2.50
Oyster Bay High 311 ORANGE CTENIDIA SN6-16 1 mL 80-100 7/10/19 2 7/12/19 1 144 174 25,056 2.87
Oyster Bay High 312 ORANGE CTENIDIA SN6-17 1 mL 80-100 7/10/19 2 7/15/19 2 144 97.4 14,026 5.13
Oyster Bay High 313 ORANGE CTENIDIA SN6-18 1 mL Not recorded 7/10/19 3 7/15/19 2 144 76.2 10,973 6.56
Oyster Bay High 314 ORANGE CTENIDIA SN6-19 1 mL Not recorded 7/10/19 3 7/15/19 2 149 49.6 7,390 10.08
Oyster Bay High 315 ORANGE CTENIDIA SN6-20 1 mL 70-100 7/10/19 4 7/15/19 2 149 TOO HIGH >28,000 #VALUE!
Oyster Bay High 316 ORANGE CTENIDIA SN6-21 1 mL 100 + 7/12/19 4 7/16/19 3 149 TOO HIGH >28,000 #VALUE!
Oyster Bay High 317 ORANGE CTENIDIA SN6-22 1 mL 70-90 7/12/19 5 7/16/19 3 149 TOO HIGH >28,000 #VALUE!
Oyster Bay High 318 ORANGE CTENIDIA SN6-23 1 mL 60-80 7/12/19 6 7/16/19 4 149 182 27,118 2.75
Oyster Bay High 319 ORANGE CTENIDIA SN6-24 1 mL 20-40 7/12/19 7 7/16/19 4 149 61.8 9,208 8.09
Oyster Bay Ambient 321 YELLOW CTENIDIA SN6-25 1 mL 60-80 7/10/19 2 7/12/19 1 144 188 27,072 2.66
Oyster Bay Ambient 322 YELLOW CTENIDIA SN6-26 1 mL 20-40 7/10/19 2 7/15/19 2 144 63.4 9,130 7.89
Oyster Bay Ambient 323 YELLOW CTENIDIA SN6-27 1 mL 20-40 7/10/19 3 7/15/19 2 144 110 15,840 4.55
Oyster Bay Ambient 324 YELLOW CTENIDIA SN6-28 1 mL 40-60 7/10/19 3 7/15/19 2 149 164 24,436 3.05
Oyster Bay Ambient 325 YELLOW CTENIDIA SN6-29 1 mL 70-100 7/10/19 5 7/15/19 2 149 200 29,800 2.50
Oyster Bay Ambient 326 YELLOW CTENIDIA SN6-30 1 mL 70-100 7/12/19 5 7/16/19 3 149 168 25,032 2.98
Oyster Bay Ambient 327 YELLOW CTENIDIA SN6-31 1 mL 40-60 7/12/19 6 7/16/19 3 149 94.6 14,095 5.29
Oyster Bay Ambient 328 YELLOW CTENIDIA SN6-32 1 mL 50-70 7/12/19 6 7/16/19 4 149 176 26,224 2.84
Oyster Bay Ambient 329 YELLOW CTENIDIA SN6-33 1 mL 70-100 7/12/19 7 7/16/19 4 149 150 22,350 3.33
Fidalgo Bay High 331 GREEN CTENIDIA NF6-16 1 mL 40-60 7/10/19 2 7/12/19 1 144 56.4 8,122 8.87
Fidalgo Bay High 332 GREEN CTENIDIA NF6-17 1 mL 10-40 7/10/19 2 7/15/19 2 144 63.4 9,130 7.89
Fidalgo Bay High 333 GREEN CTENIDIA NF6-18 1 mL 40-60 7/10/19 3 7/15/19 2 144 82.8 11,923 6.04
Fidalgo Bay High 334 GREEN CTENIDIA NF6-19 1 mL 30-50 7/10/19 3 7/15/19 2 149 66.2 9,864 7.55
Fidalgo Bay High 335 GREEN CTENIDIA NF6-20 1 mL 90-110 7/10/19 4 7/15/19 2 149 TOO HIGH >28,000 #VALUE!
Fidalgo Bay High 336 GREEN CTENIDIA NF6-21 1 mL 90-110 7/12/19 4 7/16/19 3 149 130 19,370 3.85
Fidalgo Bay High 337 GREEN CTENIDIA NF6-22 1 mL 100 + 7/12/19 6 7/16/19 3 149 198 29,502 2.53
Fidalgo Bay High 338 GREEN CTENIDIA NF6-23 1 mL 20-40 7/12/19 6 7/16/19 4 149 83.8 12,486 5.97
Fidalgo Bay High 339 GREEN CTENIDIA NF6-24 1 mL 10-30 7/12/19 7 7/16/19 4 149 78.6 11,711 6.36
Fidalgo Bay Ambient 341 BLUE CTENIDIA NF6-25 1 mL 20-50 7/10/19 2 7/12/19 1 144 92.4 13,306 5.41
Fidalgo Bay Ambient 342 BLUE CTENIDIA NF6-26 1 mL 80-100 7/10/19 3 7/15/19 2 144 178 25,632 2.81
Fidalgo Bay Ambient 343 BLUE CTENIDIA NF6-27 1 mL 50-70 7/10/19 3 7/15/19 2 144 146 21,024 3.42
Fidalgo Bay Ambient 344 BLUE CTENIDIA NF6-28 1 mL 40-70 7/10/19 5 7/15/19 2 149 28.4 4,232 17.61
Fidalgo Bay Ambient 345 BLUE CTENIDIA NF6-29 1 mL 70-100 7/10/19 5 7/15/19 2 149 TOO HIGH >28,000 #VALUE!
Fidalgo Bay Ambient 346 BLUE CTENIDIA NF6-30 1 mL 20-40 7/12/19 5 7/16/19 3 149 74 11,026 6.76
Fidalgo Bay Ambient 347 BLUE CTENIDIA NF6-31 1 mL 10-30 7/12/19 6 7/16/19 3 149 89.8 13,380 5.57
Fidalgo Bay Ambient 348 BLUE CTENIDIA NF6-32 1 mL 50-70 7/12/19 6 7/16/19 4 149 51.4 7,659 9.73
Fidalgo Bay Ambient 349 BLUE CTENIDIA NF6-33 1 mL 10-40 7/12/19 7 7/16/19 4 149 55.4 8,255 9.03

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