qPCR – Check for RLP Presence in Samples from 20120525

Performed qPCR on the samples collected during the differential centrifugation procedure on 20120525 to potentially “enrich” the samples for RLP, while eliminating background abalone tissue that would improve high-throughput sequencing results. Used a 3e6 RLP plasmid dilution (from DATE) as a positive control. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

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.

qPCR – Filter Extraction Method Comparison (from 20111107) and Differential Centrifugation Samples (from 20111116)

Performed qPCR on the three different filter extractions from 20111107. Also performed qPCR on the two samples from the differential centrifugation from 20111116. All qPCR reactions also contained an internal positive control (IPC) from the SPUD assay to assess inhibition. Additionally, ran a standard curve using Lisa’s Withering Syndrome standards 1x – 8x. I do not know the date/source of these standards, although Lisa says they were prepared by Nate. All samples and standards were run in duplicate. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the Results (see below).

Results:

qPCR Data File (BioRad CFX)

qPCR Report (PDF)

Filter Extractions

  • DNeasy Kit produced the highest signal (i.e. lowest Cq) of the three. Will use this method for future filter extractions.

  • No inhibition detected in any samples

Differential Centrifugation Fractions

  • Both fractions amplified!

  • The “Top” fraction had higher amplification than the bottom fraction

  • No inhibition detected in either sample, despite them being raw, “dirty” samples

Standard Curve

  • The 1x standard is screwy and throws off the entire curve, if left in the analysis

  • The rest of the curve looks fabulous, with proper spacing between Cq and subsequent dilutions (~3.2 Cq; assuming each standard was a 10-fold dilution from the previous).

UPDATED 20121024 – Modified data file (and subsequently the qPCR Report) to have a baseline threshold of 400 and cycles to analyze 41 to match existing conditions used for the withering syndrome qPCR assay validation.

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.