qPCR – CDFW White Abalone Samples (RLOv DNA helicase)

The samples that CDFW sent us earlier were previously checked for RLO presence with the withering syndrome qPCR assay.

Standard curve was from 20151106.

All samples were run in duplicate.

Master mix calcs are here; since I ran these with the other samples, the master mix used was part of the other project indicated in the spreadsheet (Google Sheet): 20170420 – qPCR RLOv DNA Helicase

Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Baseline threshold was manually set to 580.5, as previously determined.

Results:

qPCR Report (PDF): Sam_2017-04-20 07-50-18_CC009827.pdf
qPCR Data File (CFX): Sam_2017-04-20 07-50-18_CC009827.pcrd

Standard curve looks good and all samples provided come up positive for RLOv DNA helicase.

I’ve compiled the raw data of both the WSN qPCR and this in this Google Sheet: 20170420_CDFW_White_Ab_qPCR_summary

Here’s a summary table of the results (copy numbers are mean copies from qPCR replicates):

SAMPLE RLOV DNA HELICASE (COPIES) WSN1 (COPIES)
SF16-76_DG-1  165318.58 169.25
 SF16-76_DG-2  47839.81  20.70
 SF16-76_PE-1  1036697.17 633.75
 SF16-76_PE-2  46763.60  296.83
 SF17-17  117.29  2.16

NOTE: The WSN1 copies for SF17-17 is below the accepted detection limit of the qPCR assay (i.e. < 3 copies).

Will share my notebooks and spreadsheet with Blythe at CDFW.

Amplification Plots

Green = Standard Curve

Blue = Samples

Red = No template control

 

 

Sample ID – Black Abalone DNA for RLOv qPCRs

Carolyn & Stan Langevin have agreed that the DNA helicase qPCR should be tested on 10 black abalone DNA extractions that fall into multiple levels of the Friedman Lab’s withering syndrome histology scoring.

Downloaded the (Google Sheet) Black Abalone: Expt 1 – WS & Phage as a CSV file. After downloading, I renamed the file (Black_Abalone.csv) to facilitate easier usage in the following steps.

Created a sqlite database using GitBash for Windows:
Change to directory where file is located:

$cd Downloads

Start sqlite:

$sqlite3

Tell sqlite that the field separator will be commas (i.e. CSV file):

sqlite>.separator ","

Import the CSV file and provide a name for the resulting database:

sqlite>.import Black_Abalone.csv BlackAbs

Set output display mode to column for easier reading:

sqlite>.mode column

Set output display to include column headers:

sqlite>.headers on

 

To select all the samples that have scores of 0 in both PE and DG RLO fields (screen cap does not show entire output list):

 

To select all the samples that have scores of 1 in both PE and DG RLO fields:

 

To select all the samples that have scores of 2 in both PE and DG RLO fields:

 

Here are the full set of results in a table

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
06:5-03 06:5-35A 06:5-31
06:5-04 06:50-08 06:5-32B
06:5-08 06:50-10 06:6-46
06:5-09 06:6-32 06:6-49
06:5-10 06:6-39 08:3-05
06:5-11 06:6-42 08:3-07
06:5-14 06:6-44 08:3-15
06:5-16 06:6-52 08:3-16
06:5-18 06:6-54
06:5-20 07:12-18
06:5-21 08:3-08
06:5-22 08:3-10
06:5-24
06:5-30
06:50-04
06:50-05
06:50-11
06:50-12
06:50-13
06:50-15
06:50-16
06:6-01
06:6-02
06:6-03
06:6-05
06:6-08
06:6-11
06:6-12
06:6-13
06:6-15
06:6-16
06:6-17
06:6-18
06:6-20
06:6-21
06:6-22
06:6-23
06:6-24
06:6-25
06:6-26
06:6-27
06:6-28
07:12-01
07:12-02
07:12-03
07:12-04
07:12-05
07:12-06
07:12-07
07:12-09
07:12-10
07:12-13
07:12-19
08:3-01
08:3-02
08:3-03
08:3-04
08:3-13
08:4-01
08:4-02
08:4-03
08:4-04
08:4-05
08:4-06
08:4-07
08:4-08
08:4-09
08:4-10
08:4-11
08:4-12
08:4-13
08:4-14
08:4-15
08:4-16
08:4-17
08:4-18
08:4-19
08:4-20
08:4-21
08:4-22
08:4-23
08:4-24
08:4-25
08:5-06

Will select just 10 of those in the RLO/RLOv 0 column for use in qPCR.

I was able to track down the boxes where are these DNAs were stored (see images below).

Boxes that were not labeled with accession numbers of the samples contained therein are now labeled.

Boxes that contained samples that belonged in other boxers were transferred to the appropriate box.

All boxes were located, and returned, to the big -20C in 240 on Lisa’s shelf.

Abalone Sampling – Post-esophagus & Digestive Gland

Helped Ava sample 162 red abalone (Haliotis rufescens).

Accession numbers 15:11-1 through 15:11-162

  • Animals were weighed.
  • Took one post-esophagus sample and one digestive gland sample for histology, placed in histology cassette; three animals per cassette.
  • Took one post-esophagus sample in 95% ethanol for qPCR
  • Shells were labeled and retained for post-sampling weighing/measurements.

Abalone Sampling – Post-esophagus & Digestive Gland

Helped Ava sample 74 red abalone (Haliotis rufescens).

Accession numbers 15:10-1 through 15:10-74

  • Animals were weighed.

  • Took one post-esophagus sample and one digestive gland sample for histology, placed in histology cassette; three animals per cassette.

  • Took one post-esophagus sample in 95% ethanol for qPCR

  • Shells were labeled and retained for post-sampling weighing/measurements.

Abalone Sampling – Post-esophageal & Digestive Gland Tissues

Ava sent up abalone for sampling from California for tissue sampling. Here’s the summary of how things went.

  • Animal temps @ -8.0C when opened at lab
  •  ~120 animals sampled
  •  Only 1 dead animal found- Additional dead abalone included in shipment were NOT sampled; too dead
  • Started @ ~11AM, finished at 5:15PM (3 people dissecting, 1 person weighing)
  • All mesh bags were saved and are in a 10% bleach solution over night in sink in 236
  • Histo cassettes put in Davidson’s over night; need to be transferred to 70% EtOH tomorrow
  • Histo cassette layout: animal #1 upper left, animal #2 upper right, animal #3 lower left
  • Tubes for 15:9 sampling have labels applied
  • Histo cassettes for 15:9 sampling have NOT been labelled
  • Empty shells have been labelled and saved; will need to be weighed/measured later

 

 

RNA Isolation – Post-Esophagus Tissue from 20120525

Isolated RNA from the fractions collected on 20120525 during the differential centrifugation procedure: Gradient Top, Gradient Junk, Gradient Bottom, Hot PE Pellet, 12:6-1 (Control PE). Samples were isolated with in 1mL TriReagent according to protocol. Samples were resuspended in 0.1% DEPC-H2O, spec’d on the Roberts’ Lab NanoDrop1000 and stored @ -80C.

Results:

The second “Gradient Junk” sample on the spec results above is actually the control 12:6-1 sample.

Overall, the sample quality (based on the OD260/280) looks poor. However, this is NOT uncommon for this tissue type (PE gland) from abalone. Yields from the control sample (12:6-1) and the Hot PE Pellet are excellent. The yields from the gradient samples are low and won’t provide enough sample for high-throughput sequencing (for which this procedure was performed). Will discuss with Steven, Carolyn and Lisa for how we want to proceed.

Differential Centrifugation – Isolation of Ricketssia from Red Abalone Post-Esophegus Tissue

Post-esphagus (PE) tissue was isolated from one control abalone (12:6-1; 0.077g PE) and three “hot” abalone (11:8-8, -9, -10; 0.1606g PE, 0.126g PE, 0.1205g PE) by Lisa. Control abalone PE was homogenized in 0.5mL TriReagent and stored @ -80C. The three “hot” abalone PE were individually homogenized in ice cold 5mL of 1x Tris Sucrose Buffer (TSB). pH = 7.4 until the entire tissue was fully homogenized, including the difficult connective tissue.

Samples were transferred to 15mL conical tubes and spun at 250g for 10mins @ 4C. The supe was transferred to a 30% Percoll-TSB gradient. The pellet was placed into 1mL TriReagent, vortexed and stored @ -80C (Hot PE Pellet). 25uL from the pellet and the supe were saved for qPCR analysis.

The gradient was spun at 25000g for 2hrs at 4C in a Sorvall T21 centrifuge in a SL-50T rotor with the “SoftSpin” setting on and the brake turned off.

Below is a link to a slide show of the sample at various stages of preparation, including images of what the gradient looked like after the final spin.

De-paraffinization – Abalone PE from OTC15-250 for DNase Tests

Performed de-paraffinization according to the Friedman Lab protocol on 0.0185g of thinly sliced tissue from the tissue above in a single 5mL snap-cap tube. Briefly, tissue was treated as follows:

  • SafeClear II – 3mins x 3
  • 100% EtOH – 3mins x 2
  • 95% EtOH – 3mins x 2
  • 80% EtOH – 3mins x 2
  • 70% EtOH – 3mins x 2
  • 50% EtOH – 3mins x 2

Sample was left in 50% EtOH at RT until ready for further processing.

Differential Centrifugation – Isolation of Rickettsia from Red Abalone Post-Esophagus

Post-esophagus tissue was dissected and weighed (0.423g) by Lisa from a large Red Abalone (###) that had been previously infected with Withering Syndrome. Tissue was homogenized in ice cold 3mL Tris-Sucrose buffer (TSB; ) with a glass, 7mL homogenizer on ice. Homogenate was transferred to a 15mL conical tube. Homogenizer components were rinsed with 1mL TSB and transferred to the same 15mL conical tube. The homogenate was spun 10mins, 250g, 4C. Supe (dark brown, milky) was evenly split onto two Percol gradients (35mL; made with TSB); 30% and 50%. Gradients were spun 1hr, 25,000g, 4C in a Sorval T-21 centrifuge with a SL-50T rotor using the “SoftSpin” feature and the brake off.

Results:

Gradients didn’t show any distinct layers/bands beside a layer that seemed to reside on top of each of the Percoll gradients, as though the sample never entered the gradient. This layer seemed to contain larger cell debris which was brown in color and a hazy “sub” layer. However, there was also a small amount of cellular debris suspended (not pelleted) near the very bottom of each tube.

The top and bottom fractions were collected separately from each of the gradients. The top fractions were combined and the bottom fractions were combined. These fractions were transferred to new high-speed centrifuge tubes. The samples were washed with 1x PBS (Lisa’s recipe?) by filling each tube with 1x PBS and inverting numerous times. The tubes were then centrifuged @ 20,000g, 4C, 30mins in the same centrifuge and rotor described above. Supe was removed and the pellets (which were mostly loose) were resuspended in ~1.2mL of 1x PBS. These samples were transferred to snap cap tubes and stored temporarily @ 4C.

The pellet from the top fractions contained a significant amount of insoluble, cellular debris which settles relatively quickly to the bottom of the tube, leaving a brown, turbid supernatant. The pellet from the bottom fractions is mostly brown and turbid, with virtually no visible cellular debris.

Differential Centrifugation – Isolation of Rickettsia from Red Abalone Post-Esophagus

Post-esophagus tissue was dissected and weighed (1.9g) by Lisa from a large Red Abalone (11-8-2) that had been previously infected with Withering Syndrome. Tissue was homogenized in ice cold 3mL Tris-Sucrose buffer (TSB; ) with a glass, 7mL homogenizer on ice. Homogenate was transferred to a 15mL conical tube. Homogenizer components were rinsed with 1mL TSB and transferred to the same 15mL conical tube. The homogenate was spun 10mins, 250g, 4C. Supe (dark brown, milky) was evenly split onto two Percol gradients (made with TSB); 30% and 50%. Gradients were spun 1hr, 25,000g, 4C in a Sorval T-21 centrifuge with a SL-50T rotor using the “SoftSpin” feature.

Results:

Tubes were destroyed during spinning! Tubes were Beckman-Coulter Quick-Seal RoundTop Centifuge Tubes (Cat#: 244236). Called Beckman-Coulter. Turns out that these tubes require a special heat-sealing device AND specialized aluminum adapters for use in the rotors. Will order “normal” high speed centrifuge tubes and repeat experiment.