Tag Archives: RLOv

qPCR – RLOv DNA Helicase 2010 Water Filter DNA

Since we have a working qPCR for detecting the withering syndrome bacteriophage (RLOv), Carolyn wanted to see how detection/quantification compared to withering syndrome detection/quantification on water samples collected from various farms and the nearest wild abalone site.

DNA samples used were extractions from water filters collected for the Ab Endo Project in 2010.

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151228 – qPCR RLOv 2010 H2O Filters

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

qPCR LABEL FULL DESCRIPTION FARM/WILD SITE NEAREST FARM/WILD SITE
painted-SCI_B Painted Cave SCI B Wild The Cultured Abalone
painted-SCI_A Painted Cave SCI A Wild The Cultured Abalone
prison-SCI_B Prisoner’s SCI B Wild The Cultured Abalone
prison-SCI_A Prisoner’s SCI A Wild The Cultured Abalone
TCA_out_East_A The Cultured Abalone Outfall East A Farm Santa Cruz Islands
TCA_out_East_B The Cultured Abalone Outfall East B Farm Santa Cruz Islands
TCA_out_West_A The Cultured Abalone Outfall West A Farm Santa Cruz Islands
TCA_out_West_B The Cultured Abalone Outfall West B Farm Santa Cruz Islands
TAF_ND_A1 The Abalone Farm North Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A1 The Abalone Farm South Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A2 The Abalone Farm South Drain A2 Farm Pt. Sierra Nevada/Rancho Marina
RM_A Rancho Marina A Wild The Abalone Farm
RM_B Rancho Marina B Wild The Abalone Farm
AmA_Drain_A1 American Abalone Drain A1 Farm Carmel
AmA_Drain_A2 American Abalone Drain A2 Farm Carmel
AmA_Drain_B1 American Abalone Drain B1 Farm Carmel
AmA_Drain_B2 American Abalone Drain B2 Farm Carmel
PSN_0M_A Pt. Sierra Nevada 0M A Wild The Abalone Farm
PSN_0M_B Pt. Sierra Nevada 0M B Wild The Abalone Farm
CP_0M_A1 Carmel 0M A1 Wild American Abalone
CP_0M_B Carmel 0M B Wild American Abalone
CP_0M_A Carmel 0M A Wild American Abalone

Results:
qPCR Report (PDF): Sam_2015-12-28 11-20-16_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-28 11-20-16_CC009827.pcrd

Overall, data looks good. Will enter copy numbers into the Ab Endo master sheet for later analysis (Google Sheet): Ab Endo Samples

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qPCR – RLOv DNA Helicase Standard Curve Check Repeat

Yesterday’s check of the RLOv DNA helicase standard curve using a freshly made 3 copy sample didn’t look perfect, so I’m re-running to see if things will tighten up a bit.

Repeated yesterday’s run with no changes.

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151223 – qPCR RLOv DNA Helicase Curve Check

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

Results:
qPCR Report (PDF): Sam_2015-12-24 11-02-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-24 11-02-59_CC009827.pcrd

 

Overall, things are looking better than yesterday’s run: better reps, better efficiency and better R^2. Will move forward with beginning to validate this qPCR assay, as well as use for some other sample analysis that Carolyn has in mind (comparing RLOv vs. WS levels in abalone collected from wild sites in California).

 

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qPCR – RLOv DNA Helicase Standard Curve Check

Since the standard curve for this assay was a bit wonky at the low copy number end the last time I ran it, I made a fresh 1:10 dilution of the 3 copies component of the curve (100μL of 30 copy sample in 900μL TE).

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151223 – qPCR RLOv DNA Helicase Curve Check

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

Results:
qPCR Report (PDF): Sam_2015-12-23 13-57-01_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-23 13-57-01_CC009827.pcrd

 

The efficiency & R^2 values look pretty solid, but the spacing between the 300 copy (Cq ~ 32 in the amplification plot) and the 30 copy (Cq ~ 34 in the amplification plot) samples is a bit too tight for my liking. Additionally, the reps for the 3 copy sample are very poor.

Will repeat to see if I can tighten things up…

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In-situ Hybridization (ISH) – RLOv Membrane Gene 1, Tail Fiber Gene: Day 3

All washes/rinses were performed in cylindrical glass slide incubators at room temp (30mL):

  • Slides were briefly rinsed in dH2O three times.
  • Slides were counter stained with 0.05% aqueous Bismark Brown Y for 3mins.
  • Slides were briefly rinsed in dH2O, then 70% EtOH, then 100% EtOH.
  • Slides were air-dried in the fume hood.
  • Coverslips were added to each slide with three drops of Permount.
  • Permount was allowed to dry O/N at RT.

Images were captured using Nikon BR Essentials.

The same section of each slide (within an accession number set) was captured at 4x, 10x, and 20x magnifications for comparisons. Auto white balance adjustment was the only image manipulation performed. All images (see Results below) are as they were captured by the software.

Results:

Quick summary: Both probes appear to be functional! With that being the case, I will proceed to run ISH on black abalone samples (these test ISHs were with red abalone) for the proper assessment of RLOv localization.

All images are here (Dropbox): 20151204_ISH_RLOv

Tryptic images of 10x magnifications are presented below showing the H&E staining, negative control and RLOv probe.

ISH staining is expected to appear as dark brown staining.


08:1-7 (RLOv)

H&E – RLO inclusions are seen as the deep purple oblong structures.

Negative Control – RLO inclusions exhibit no staining and appear as oblong empty regions. These regions also no have any apparent cell wall/membrane around them. This is in contrast to the two other accession groups (08:1-12 & 08:1-15).

RLOv tail fiber – Staining is noticeable surrounding the RLO inclusion locations, but not within the inclusions. The staining is similar to the 08:1-12 negative control. So, it’s difficult to say if the staining in this sample is binding to its intended target (RLOv tail fiber) or if the difference seen is simply due to the particular section of this tissue.

RLOv membrane gene 1 – Not shown due to this region of tissue not being present on the slide.

 


 

08:1-12 (RLO CLASSIC)

H&E – RLO inclusions are seen as the deep purple oblong structures.

Negative Control – RLO inclusions appear similar to air bubbles, with no staining within them.

RLOv probes – Both probes stain within the RLO inclusions.


 

08:1-15 (RLO STIPPLED)

H&E – RLO inclusions are seen as light purple bulbous structures.

Negative Control – RLO inclusions are actually stained brown and are very noticeable. This is not expected.

RLOv membrane gene 1 - The staining is in the same locations as the negative control RLO inclusions. Intensity-wise, the staining seen from this probe is not much different than the negative control. However, the way the staining appears within the inclusions is different than the negative control. Not sure if this indicative of the probe working or if the different appearance is due to difference in tissue sections.

RLOv tail fiber – The staining is in the same locations as the negative control RLO inclusions. However, the intensity of the staining with the RLOv tail fiber probe is a much deeper brown, suggesting that the probe is binding within the RLO inclusions.

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In-situ Hybridization (ISH) – RLOv Membrane Gene 1, Tail Fiber Gene: Day 2

All washes/rinses were performed in cyclindrical glass slide incubators (30mL):

STRINGENCY WASHES

  • All SSC washes were pre-heated to appropriate temps before proceeding
  • Hybridization solution was discarded and slides rinsed for ~30mins in 2x SSC.
  • Cover slips were removed.
  • Slides were washed twice in 2x SSC 15mins @ 40C.
  • Slides were washed three times in 1x SSC 15mins @ 40C.
  • Slides were washed once in 0.5x SSC 15mins @ 40C.
  • Tissue was equilibrated in Buffer 1 (100mM Tris-HCl, 10mM NaCl, pH = 7.5) 10mins @ RT.
  • Tissues were blocked with Blocking Buffer (Buffer 1 + 2% sheep serum + 0.3% Triton-X 100) for 1hr @ RT (500uL on each slide w/cover slips – sitting flat on bench top).

DETECTION

  • Antibody solution (Anti-Digoxigenin-AP Fab fragments [Roche - Exp Nov. 2010]) – Diluted anti-DIG 1:1000 in Blocking Buffer. Anti-DIG is stored @ 4C in FSH 240 on Lisa’s shelf.
  • Added 1mL of antibody solution to each slide and incubated without a cover slip for 2hrs @ RT – sitting flat on bench top.
  • Rinsed slides with Buffer 1 for 10mins, two times.
  • Rinsed slides with Buffer 2 (100mM tris-HCl, 100mM NaCl, 50mM MgCl2; pH = 9.5) for 10mins.
  • Incubated slides in substrate solution (30mL Buffer 2 + 135uL NBT [Roch - Exp Feb 2010 - 4-nitro blue tetrazolium] + 75uL BCIP [Roche Exp Nov 2010 - 5-Bromo-4-chloro-3-indolyl phosphate]) O/N @ RT in the dark. Both NBT & BCIP are stored @ -20C in FSH 236 in the “ISH Reagents” box.
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In-situ Hybridization (ISH) – RLOv Membrane Genes 1 & 2, Tail Fiber Gene: Day 1

To test out the viability of these RLOv ISH probes (from 20151109) and not waste black abalone slides if this doesn’t work, I selected three unstained red abalone post-esophagus sections:

RLO – NO PHAGE

  • 08:1-12-2
  • 08:1-12-3
  • 08:1-12-4

RLOv

  • 08:1-7-7
  • 08:1-7-8
  • 08:1-7-9

RLO STIPPLED – NO PHAGE

  • 08:1-15-7
  • 08:1-15-8
  • 08:1-15-9

All slides were processed in a single, horizontal glass slide incubator (200mL), unless otherwise noted.

All steps were conducted at room temperature (RT), unless otherwise noted.

DEPARAFFINIZATION & REHYDRATION

  • All slides were deparaffinized with three changes of xylene (SafeClear II; Fisher) for 10mins each.
  • Slides were hydrated with a graded ethanol series (100%, 100%, 80%, 70%, 50%) for 3mins each.
  • Slides were rinsed with molecular grade H2O.

PREHYBRIDIZATION

  • Tissue sections were equilibrated in Tris Buffer (0.2M Tris-HCl, 2.0mM CaCl, pH = 7.2) for 5mins.
  • Tissues were permeabilized for 1.5hrs in preheated 50ug/mL Proteinase K (Qiagen) in Tris Buffer @ 56C.
  • Slides were rinsed with 1x PBS three times, 10mins each.
  • Slides were incubated 30mins in 30mL Prehybridization Buffer (50% deionized formamide, 4x SSC) @ 53C in a cylindrical glass slide incubator due to limited volume of deionized formamide available:

  • Prepared probes by boiling 3mins and immediately incubating in ice water bath for 30mins.
  • Slides were rinsed with 2x SSC and air dried for 5mins.
  • Probes were diluted 1:300 in 1000uL of Prehybridization Buffer. All three negative control probes (indicated by “-C” in subsequent labeling) were combined into a single dilution.

NOTE: RLOv Membrane Gene 2 probe was ruined because boiling water got into the tube during denaturation. This didn’t happen to any of the other tubes that were all boiled at the same time. Not sure what happened. However, this may have worked out OK because I did not pull enough slides to accomodate the negative control probes. So, now that I’m not able to test three probes, I can use the negative control probes!

HYRBIDIZATION

  • 300uL of probe solutions and cover slip were added to the following slides:

  • The three groups of slides were placed into separate slide cases and a 1mL of Prehybridization Buffer was added to each case (to maintain high humidity during incubation).
  • The cases were incubated on their sides O/N @ 53C.
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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.

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Data Management – Black Abalone Histology Scores

As part of the qPCR validation for the withering syndrome phage (RLOv) project, I needed to identify (and, eventually locate) samples that are infected with varying levels of RLOv. This is probably the most time consuming aspect of the project.

I found the histology scoring sheets and added them to an existing Google Sheet that Lisa had partially completed a few years ago: Black Abalone: Expt 1 – WS & Phage

To save time, I only entered the scores into the spreadsheet and did not enter any extra info (like Sex or Coccidia).

Having this data in a single, digital format will allow me to sort the data, to quickly & easily select the appropriate samples with varying levels of RLOv (categorized as “New” on the sheet).

Here are links to pics of the histology scoring sheets for reference:

Next up will be to actually track down the physical samples. This will be a bit of a daunting task…

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PCR – RLOv In-situ Hybridization (ISH) Probes

Ran probe-labeling PCRs to use in in-situ hybridization (ISH) using the PCR DIG Probe Sysnthesis Kit (Roche). Generated PCR probes for using the following BamHI-linearized plasmids:

  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_tail_fiber

The Roche protocol recommends using only 10pg of plasmid DNA for probe labelling. As such, all three probes were diluted 1:10,000. A 1:1000 (999μL H2O + 1μL of plasmid) was made first. Then a 1:10 dilution was made (90μL H2O + 10μL from 1:1000 dilution of plasmid).

Additionally, I ran half reactions to conserve kit components. Roche recommends 50μL reactions; I ran 25μL and scaled all components appropriately.

All reactions were set up on ice and run in 0.2mL strip-cap PCR tubes.

Reaction calculations are here (Google Sheet): 20151109 – RLOv ISH Probe PCRs

Cycling params:

  1. 95C – 5mins
  2. 95C – 15s
  3. 55C – 15s
  4. 72C – 30s
  5. Go to Step 2, repeat 39 times.
  6. 72C – 10mins

After the PCR, 5μL of each reaction was run on a gel.

Results:

Hyperladder I (Bioline)

PCR DIG probe labelling products run on 1.1% agarose 1x TBE gel stained w/EtBr. A ‘+’ indicates DIG reaction, while a ‘-‘ indicates no DIG in reaction.

Two reactions were run for each plasmid: one with the DIG label (indicated by a ‘+’) and one without (indicated by a ‘-‘). If the labeling was successful, the PCR products from those reactions containing DIG will be larger (i.e. migrate slower) than those without. That is exactly what we see in each of the three potential ISH targets.

So, we now have three ISH probes ready for action! Will proceed with making fresh ISH buffers and ISH.

Probes were transferred to 0.5mL snap cap tubes and stored in my -20C box.

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Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20

 

Digestion Master Mix

REAGENT SINGLE REACTION (μL) x 5.5 (μL)
Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.

Results:

Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).

 

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