Kaitlyn’s notebook: Geoduck hemolymph qPCR

qPCR was performed on samples with successful RNA extractions (by me and Sam) and cDNA ((by me and Sam) with primers designed by Sam according to the Roberts lab SOP. Here is a list of who extracted and RTed which sample:

  • Kaitlyn: 1, 2, 19, 27, 59, 62, 66, 11/15 Chewy, 11/15 Star, 11/21 Chewy, 11/21 Star
  • Sam:  57, 61, 39, 31

Here is my qPCR master mix calculations. Sam previously ran a qPCR using the above samples. All qPCR reactions were run in duplicate.

Conclusions

I achieved the same results as the previous qPCR run by Sam on his samples. A total of 2 known males and 2 known females amplified. Males had less VTG present based on the CF value (later = less amplification) compared to females. The later stage female had about 5 fold less VTG present. However, the amplification in other samples did not reach thresholds or occur.

The primers were designed base don an older geoduck genome, therefore we want to redesign primers with the most recent genome. We want to explore all selected targets identified in Emma’s paper to explore other possible proteins that may be a better match.

Kaitlyn’s notebook: Geoduck hemolymph Reverse transcription (cDNA)

I made cDNA from samples I had successfully extracted RNA from (1, 2, 19, 27, 59, 62, 66, 11/15 Chewy, 11/15 Star, 11/21 Chewy, 11/21 Star).

Reverse transcription was performed using 50ng of each sample with M-MLV Reverse Transcriptase from Promega accroding to the Roberts Lab SOP. Calculations can be found here. A 1:1000 dilution of primer was made to increase pipetting volume.

Samples were initially heat to 45C for 5 min for denaturation instead of 70C, however the incubation was redone immediately after at 70C. This would not impact the cDNA because the step only serves to denature the RNA to maximize primer binding.

Samples are stored in the -20C fridge in 209 in 20190107 Kaitlyn’s Box. Additionally, the hemolymph cDNA created by Sam is also located in the same box.

 

Kaitlyn’s notebook: Hemolymph RNA extractions 2

RNA was isolated with a Quick-DNA/RNA Microprep Plus Kit by ZymoResearch. The samples are stored in a box in the -80C freezer in 3, 3, 2, labelled “RNA isolations; geoduck 12/17”. Isolations were done according according to the manufacturer’s protocol on the following samples:

G-32 H
G-47 H
G-53 H
G-59 H
G-62 H
G-63 H
G-29 H
G-19 H
G-22 H
G-64 H
G-66 H
G-40 H
G-58 H
G-50 H

For hemolymph, 600ul of sample was taken and 2400ul of lysis buffer was added for prep. If 600ul of sample was not available, all of the sample was taken. The on-column DNase step was done, and the elution volume was 15ul.

Several columns were getting clogged from tissue in the sample.

Samples were quantified with the hsRNA Assay for Qubit according to manufacturer’s protocol. 1ul of sample was used and 199ul of working solution was used in each assay tube. All values were too low for quantification which was lickely caused by overloading the column, column clogging, and/or unknown technical error.

Kaitlyn’s notebook: Geoduck RNA extraction

RNA was isolated with a Quick-DNA/RNA Microprep Plus Kit by ZymoResearch according to the manufacturer’s protocol from geoduck samples .

For hemolymph, 150ul of sample was taken and 600ul of lysis buffer was added for prep, except for sample 27 which contained 120ul of sample and had 48-ul of lysis buffer added. All centrifuge steps were done at 16,000 rpm.

The on-column DNase step was done, and the elution volume is 15ul.

Samples were quantified with the hsRNA Assay for Qubit according to manufacturer’s protocol. 1ul of sample was used and 199ul of working solution was used in each assay tube.

The samples are stored in a box in the -80C freezer in 3, 3, 2, labelled “RNA isolations; geoduck 12/17”.

Sample RNA (ng/ul)
Star 11/15 47.2
Chewy 11/15 92.6
Star 11/21 170
Chewy 11/21 180
1 12.5
2 33.8
40 low
62 4.2
58 low
47 low
53 low
50 low
19 4.6
66 4.2
22 low
29 low
32 low
59 100
63 low
27 134
64 low
Standard 1 38.07
Standard 2 395.24

Samples are located in:

  • Box 8,1,1 (2014): Geo- 27, 31, 29
  • Box 8,1,3 (2015): Geo-  50, 58, 62, 19, 64, 40, 59, 47, 53, 63, 22, 66.
20191217_082151

Box 3,1,1 on left and Box 8,1,3 on right separated by Star and Chewy samples. Samples 1 and 2 and Star and Chewy samples are in box 5,3,1.

Sample Geo-30 H did not exist, although a Geo-30 did but it looked like gonad so I did not extract from it. 20191217_082036.jpg

Notes:

Sample 53 was dark green.

 

Kaitlyn’s notebook: ANOVA on heath stack juveniles

I preformed an ANOVA on the lengths of the juveniles in the heath stacks at Pt.Whitney. I did a post-hoc test (Tukey HSD) to determine significance between he treatments.

length-treatment

length-treatment

homogenization

With blocking the anova gives a p=0.028 for treatment and p=0.245 for tray.

Here is the Tukey HSD p-values on the anova with blocking:

Tukey multiple comparisons of means
95% family-wise confidence level

AE-AA 0.54267725
EA-AA 0.25261761
EE-AA 0.72476397
EA-AE 0.91813748
EE-AE 0.10032636
EE-EA 0.03504769

H0_T-H0_B 0.9999666
H1_B-H0_B 0.9999930
H1_T-H0_B 0.9303406
H2_B-H0_B 0.9999448
H2_T-H0_B 0.9970459
H3_B-H0_B 0.9994060
H3_T-H0_B 0.9988241
H1_B-H0_T 0.9980676
H1_T-H0_T 0.8107845
H2_B-H0_T 0.9963141
H2_T-H0_T 0.9703042
H3_B-H0_T 0.9875827
H3_T-H0_T 0.9999998
H1_T-H1_B 0.9588824
H2_B-H1_B 1.0000000
H2_T-H1_B 0.9996848
H3_B-H1_B 0.9999842
H3_T-H1_B 0.9708530
H2_B-H1_T 0.9898375
H2_T-H1_T 0.9991863
H3_B-H1_T 0.9962770
H3_T-H1_T 0.4563048
H2_T-H2_B 0.9999849
H3_B-H2_B 0.9999999
H3_T-H2_B 0.9641553
H3_B-H2_T 0.9999998
H3_T-H2_T 0.8273796
H3_T-H3_B 0.9074722

Tank Treatment
H0_T EA
H0_B EE
H1_T AE
H1_B AA
H2_T EA
H2_B AA
H3_T AE
H3_B EE

Kaitlyn’s notebook: Quantifying geoduck histology

These are possible measurements that can be taken on the geoduck histology slides based on tools I’ve found:

Males:

  • pixel classification: acini vs connective tissue
  • average acinus size (measure widest portion of 25 acini/individual)

Females:

  • oocyte length
    • measure only round eggs with visible nucleus
  • average follicle size (measure widest potion of 25 follicles/individual)

Unfortunately I can’t use a pixel classifier for the females because the oocytes and connective tissue stain is very similar in color and there are significant white space between the tissue and in the nucleus. 

I have been using the Padilla-Gamiño micrscope (Nikon Eclipse Ni-U with the Nikon Elements BR [basic research] program). I have been exploring the program to find features useful to our histology slides. I can get the % area using the pixel classifier.

 

20190521_110608

Acini are deeply stained. Using the pixel classifier I can make these pixels red. The percent area for each phase (pixel stain) is shown to the right.

20190521_110629

Connective tissue is shown with the green pixels.

20190521_110634

A possible third phase in blue…

Alternatively, I can do just two phases-

20190521_130140

this is using the manual method (the bayes method has you pick sample pixels for each phase rather than showing a graph for the wavelengths).

However, the inability to remove the phase overlapping on the image makes it very difficult to find another spot to measure on the slide, and I surmise that at least 3-4 measurements throughout the slide should be taken to accurately determine the % area of the acini.

Other possible option are thresholding in Fiji (Fiji is just ImageJ [but updated]), color deconvolution in ImageJ (but I have not been able to find a way for the program to give me % area), or pixel classifier qith Qupath (a new addition to the program that still has soem kinks and I haven’t been able to use yet).

It is easy to take measurments and images with a scale burned onto the image so measuring oocytes, collicles and acini using either this program or ImageJ is easy! I am working on females first, and during any down time, i.e., someone else needs to use the scope, I am trying to learn how to sue Fiji, Qupath, or the Element BR program. 

Kaitlyn’s notebook: 2018 geoduck juveniles

Juveniles reared in heath stacks:

ID n Treatment Length Width
H0_T 7 E*A 10.78 7.36
H0_B 9 E*E 12.21 7.50
H1_T 15 E*A 9.71 6.75
H1_B 16 A*A 11.32 7.88
H2_T 14 E*A 9.77 6.84
H2_B 11 A*A 11.20 7.87
H3_T 14 E*A 11.35 7.65
H3_B 13 E*E 11.71 8.19

Checking on outplanted juveniles:

  • -0.76 low tide
    • We planted on very low point of the beach so it goes underwater very quickly. The ferries were very busy so I got out there alter than anticipated. I cut open a quarter of the tubes for thorough inspections. I checked another 50% without cutting open the tubes because the tide was coming in quickly, but it was difficult to see anything with the mesh shadows.
  • 3 zip-ties on each mesh tube were hard to cut off (return with more people!)
  • In the tubes:
    • 3-4 crabs (up to 3 inches big)
      • Many of the tubs had crabs the crabs were crawling in and out of
    • Cockles
      • Difficult to tell if holes are geoduck or cockles
        • Would recommend coming back with the intention of digging them up to be sure of geoduck survival
Tray Treatment Outplant Color # Holes Tube(s)
H0_T E*A Yellow 1 H12
H0_B E*E Grey 1 B1
H1_T E*A Pink 2 A1, B7
H1_B A*A Purple 1 A10
H2_T E*A Green 0
H2_B A*A Red 3 A3, A7, B11
H3_T E*A Dark blue 2 B12, D11
H3_B E*E Orange 1 B5

MVIMG_20190511_162746

MVIMG_20190511_162755.jpgMVIMG_20190511_163156