Kaitlyn’s notebook: sex/stage plots on primers

Primers by sex & stage:

Female Cq.Means by Stage:

Male Cq.Means by Stage:

• • Previous plots
  • Primer spreadsheet

 

Kaitlyn’s notebook: boxplots on sex & stage for geoduck primers

All primer pairs by sex and stage:

Cq. mean values based on sex for each primer pair tested on either just pooled or both pooled and known samples. Pooled samples are a combination of males and females across all stages.

Cq. mean values based on reproductive stage for each primer pair tested on either just pooled or both pooled and known samples. Pooled samples are a combination of males and females across all stages.

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Primers by Stage:

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Primers by Sex:

Primers tested on known samples and reported Cq.mean values.

A value of 0 was plotted for values not reported.

A value of 0 was plotted for values not reported.

Kaitlyn’s notebook: Primers next steps/goals

1. Look at product and compare to theoretical size
   • Must do visually (order based on sequence)
2. Make plots for each primer w/ pooled
   • Cq.mean
   • Melt temp
   • Melt peak height
3. Analyze Cq values
   • Sex or development differences (ANOVA?)
     • mean, melt temp and melt peak height by sex
       • And then by dev. stage
4. Make summary of performance of each primer
   • Via a table (rank performances of each primer w/ grade & notes on grade [3 columns])
     • With pooled sample
     • And known samples

Kaitlyn’s notebook: primers on known geoduck hemolymph samples

Previous qPCR test on pooled samples determined these 4 primers would be run on known samples. Ran qPCR as previously described.

Results from qPCR on known samples:

Two runs had to be done to be able to include all samples.

• • Run 1 Bio-Rad file and Run 2 Bio-Rad file
  • Run 1 .csv file and Run 2 .csv file

Results

• • contamination in no template controls (NTC)
    • unknown when contamination could have occurred
      • Water used for NTC seems unlikely since similar amplification is occurring in each sample
      • Possible contamination in primers, master mix, or DNase
        • Tris-EDTA added to lyophilized primers?
          • used 2017 DNase for RT

 

Regardless, there is non-specific amplification which also occurred in the last qPCR run on pooled samples.

The non-specific binding could instead be the presence of leftover DNA from a less effective DNase (since it was from 2017). In order to test this:

• • one RNA sample left (G38) that hasn’t been DNased
    • run qPCR on 4ng RNA from G38 (equivalent to RNA amount run in previous qPCRs)
      • Include NTC to test if primers are contaminated as well

I can dilute my cDNA 1:10 to try to make it last longer which can be seen in amplification image. I used up a substantial portion testing the 4 selected primers during these runs.

Next, Sam suggested I run pooled samples at a gradient of temperatures to identify an ideal temperature with reduced non-specific binding.

Kaitlyn’s notebook: testing new primers on geoduck hemolymph RNA

I made cDNA from previously isolated RNA. I included a pooled sample that I used to test the primers I recently designed via qPCR.

Reverse Transcriptase

RT was performed using 100ng of each sample with M-MLV Reverse Transcriptase from Promega according to the Roberts Lab SOP. All reagents were doubled to produce twice the amount of cDNA. The pooled sample was quadrupled. A 1:1000 dilution of primer was made to increase pipetting volume, and a 1:10 dilution was made for the pooled sample. Calculations can be found here.

• Pooled sample contains 61, 55F, 43B, and 19

Samples are stored in the -20C fridge in 209 in 20190107 Kaitlyn’s Box.

qPCR

Primers were tested based on the Roberts lab qPCR SOP. The primers can be found here or they can also be found in the PrimerDatabase.

There was more pooled sample loss than I expected from RT. I originally intended to use 4ul of pooled sample per primer. Instead I could only use 1ul per sample, and I spiked in 1ul of cDNA from pre-existing cDNA so that I had enough master mix avaliable for each sample.

Results

Well	Fluor	Target	Content	Sample	Cq
A01	SYBR	TIF3s6B	Unkn-01	Pooled	18.2612
A02	SYBR	TIF3s6B	Unkn-01	Pooled	18.17076
A03	SYBR	NFIP	Unkn-02	Pooled	27.42391
A04	SYBR	NFIP	Unkn-02	Pooled	26.68092
A05	SYBR	APLP	Unkn-03	Pooled	27.29234
A06	SYBR	APLP	Unkn-03	Pooled	27.19591
A07	SYBR	TIF3s10	Unkn-04	Pooled	37.34317
A08	SYBR	TIF3s10	Unkn-04	Pooled	33.8457
B01	SYBR	ECHD3	Unkn-05	Pooled	32.67729
B02	SYBR	ECHD3	Unkn-05	Pooled	33.01202
B03	SYBR	TIF3sF	Unkn-06	Pooled	21.97772
B04	SYBR	TIF3sF	Unkn-06	Pooled	22.08964
B05	SYBR	TIF3s12	Unkn-07	Pooled	32.0917
B06	SYBR	TIF3s12	Unkn-07	Pooled	32.74342
B07	SYBR	FEN1	Unkn-08	Pooled	34.8731
B08	SYBR	FEN1	Unkn-08	Pooled	NaN
C01	SYBR	SPTN1	Unkn-09	Pooled	27.68292
C02	SYBR	SPTN1	Unkn-09	Pooled	27.16297
C03	SYBR	NSF	Unkn-10	Pooled	34.57384
C04	SYBR	NSF	Unkn-10	Pooled	37.76697
C05	SYBR	TIF3s7	Unkn-11	Pooled	34.7186
C06	SYBR	TIF3s7	Unkn-11	Pooled	35.08897
C07	SYBR	TIF3s8-2	Unkn-12	Pooled	22.06597
C08	SYBR	TIF3s8-2	Unkn-12	Pooled	21.92805
D01	SYBR	TIF3s8-1	Unkn-13	Pooled	37.13184
D02	SYBR	TIF3s8-1	Unkn-13	Pooled	35.01336
D03	SYBR	TIF3s4a	Unkn-14	Pooled	NaN
D04	SYBR	TIF3s4a	Unkn-14	Pooled	NaN
D05	SYBR	GLYG	Unkn-15	Pooled	37.49771
D06	SYBR	GLYG	Unkn-15	Pooled	35.38684
D07	SYBR	RPL5	Unkn-16	Pooled	17.46911
D08	SYBR	RPL5	Unkn-16	Pooled	19.57397
E01	SYBR	GSK3B	Unkn-17	Pooled	19.45339
E02	SYBR	GSK3B	Unkn-17	Pooled	18.07122

One target primer, FEN1, only worked on one sample which may have been a technical error so it may be worth re-running. TIF3s4a, one of 9 regulatory primers, did not work on either sample, but all the others worked great!

Next…

• Choose 1-2 regulatory primers
• Retest FEN1, target, primer
• Assess known samples

Kaitlyn’s notebook: Geoduck hemolymph next steps and RNA extractions

Recap

The overall goal is to identify whether the geoduck are reproductively developed (ready to spawn), and potentially whether the geoduck are male or female, using hemolymph (which can be non-lethally collected).

So far we have…

1.Identified biomarkers for reproductive development/sex

• Emma identified shown in Fig. 6

1. Developed primers for biomarkers

• 1st primers developed by Sam
  • had variable amplification in qPCR
• 2nd set of primers designed by me

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Next Steps:

I want to test the newly developed primers, but first I need to ensure I have adequate amounts of RNA and cDNA because I now have 17 primer pairs to test.

• want to include more males/females across more reproductive stages
  • went through leftover hemolymph samples from known individuals

Identified 9 female and 7 male hemolymph samples across stages for qPCR:

Sample	Sex	Stage
19	F	2
21	F	2
23	F	2
55	F	3
31	F	4
39	F	5
37	F	6
57	F	7
61	F	7
28	M	1
27	M	2
54	M	3
43	M	4
59	M	4
62	M	5
66	M	5

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Samples for RNA extraction:

50ng RNA is needed for the RT protocol (100 ng is ideal esp. since I will use 1ul of template) so previous samples will need to be extracted again: 19, 21, 23, 55, 37, 28, 54, and 43.

Samples for RT (to make cDNA):

Samples that did not amplify previously may have had errors during RT so I will remake their RT in addition to the new samples: 19, 21, 23, 55, 37, 28, 54, 43, 39, 57, 62, and 66.

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RNA Isolation:

RNA was isolated with a Quick-DNA/RNA Microprep Plus Kit by ZymoResearch according to the manufacturer’s protocol from geoduck hemolymph samples. 300ul of sample was used and 1200ul of lysis buffer was added for sample prep. All 250ul of sample 19 was used and 1000ul of lysis buffer added instead. The RNA was NOT DNased (will need to be done before RT).

I tested the ‘whole blood’ manufacturer instructions on sample 43B; sample 43A was done normally. Sample 54 was accidentally added to the sample 55 column. I pipetted out the supernatent from the 55 column, denoted the column as 55F, and added the supernatent to the correct 54 column. Then I got a new column for the second half of 55, labelled it as 55(2) and continued the extraction.

Samples were quantified with the hsRNA Assay for Qubit according to manufacturer’s protocol. 2ul of sample and 198ul of working solution was used per assay tube. Standard 1: 92.49 RFU and Standard 2: 1881.11 RFU.

Sample	Sex	Stage	RNA (ng/ul)
19	F	2	35.8
21	F	2	18
23	F	2	low (151.89 RFU)
28	M	1	18.7
37	F	6	high (4893.75 RFU)
43A	M	4	high (3520.34 RFU)
43B	M	3	3.62
54	F	3	high (3152.78 RFU)
55F	F	7	high (3544.7 RFU)
55(2)	M	4	57

Although in RFU range, the low sample may be under ng/ul minimum and is likely not enough for RT. High samples will need to be diluted 1:2 and be requantified.

Samples are stored in a box in the -80C freezer in 3, 3, 2, labelled “RNA isolations; geoduck 12/17”. Note that blue tubes are from previous RNA isolations which were DNased, and those with ‘B’ denoted are from the second round of RNA extractions (contain 0ng RNA). The samples extracted today, which are not DNased, are in yellow tubes.

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How much cDNA should I make?

• RT protocol provides 20ul of template
  • will use 1ul of template for qPCR
    • = total of 20 samples that can be done
      • run samples in duplicate…
        • must do 2 rounds of RT to get enough cDNA for 1 round of qPCR with 17 primers
• RT calculations

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I also picked up the new primers from Biochem stores which are on the benchtop in 209.

Kaitlyn’s notebook: C. baridi hemocyte pellet RNA isolation and quantification

Samples

Isolated RNA from the following hemolymph pellet samples:

• 58
• 61
• 64
• 72
• 79
• 93
• 94
• 96
• 99
• 110
• 114
• 126
• 140
• 155
• 165
• 173
• 180
• 208
• 252
• 256

Samples 114 and 126 were previously done successfully, but due to changes in spreadsheet organization, they were repeated accidentally.

The following samples could not be found, all of which have green caps:

• 6
• 43
• 76
• 130

Samples 58 and 61 were clear (left) in contrast to the cloudy appearance of the other samples (right).

RNA Isolations

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch) according to the manufacturer’s protocol for liquids/cells in RNAlater.

• Used 35uL from each RNAlater/hemocyte slurry.
  • Except for sample 58 where all 20ul of sample were used
• Mixed with equal volume of H2O and 8x lysis buffer
• Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • Held at 4C
• Performed on-column DNase step.
• RNA was eluted in 15uL H2O

RNA quantification: HS Assay on Qubit

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.

• • For samples 58 and 61 which registered LOW:
    • Added 2ul more for for a total of 4ul to assay tube
      • still too low

Samples are currently in box in front of Rack 9 (all racks in -80C are full), but I will add to the Shellfish RNA boxes!

Kaitlyn’s notebook: Geoduck reproductive development primer design

After testing the previous primers, it was decided that the primers were not specific enough for successful sex or reproductive stage identification. I am going to design new primers with the most recent geoduck genome version of all proteins listed in Fig. 6.

I am following Sam’s Jupyter post  (downloadable here) and Github post. I am focusing on primer design and primer specificity, as the fasta file containing the 8 proteins in Fig 6. has been created.

I used jupyter notebook to annotate code and explain shell to understand new commands.

1. 1. 1. 1. Download
            • pyfaidx – splits multi-FastA into individual FastA files
            • Primer3 – performs primer design
            • EMBOSS(installation issue linked) – checks primer specificity against entire transcriptome
              • installed without X11 support (problem with X11 headers)
         2. Split FastA into multiple FastAs using pyfaidx
         3. Design primers with Primer3 (issue with code)
         4. Check specificity using EMBOSS
            • Problem with primersearch function

• • • Primer files on OWL
      
    • Geoduck primer spreadsheet
    • Roberts lab primer database

 

Kaitlyn’s notebook: 20200121 Geoduck Strip spawn @ Pt. Whitney

Shelly, Brent and I helped Matt with a strip spawn yesterday!

• Modified and tested 1000ul pipette tip and end of 50ml falcon tube for speculum
  • Inserted speculum into pedal gape
    • Used 18g needle to sample gonad
      • Put on slide and examined at 4X and 10X for sperm or egg

The modified falcon tube worked best because it was slightly larger and remained in place better when the geoduck contracted. Using a headlamp, we could actually see the gonad before taking a sample.

• Tested 2 geoduck from each tank (1, 2, 4, and 5)
  • Found 3 ripe males and a ripe female
• Shucked and removed gonad
• Cross-hatched gonad; added egg and sperm into individual tripours
• Rinsed eggs in 20um screen
  • lots of clogging from egg jelly –> used 2 screens
• Primed eggs with ~4000 mL of 50mM KCL
• Rinsed over 20um screen and added to 2L pitcher
  • Final volume of 1200ml; Cellometer estimated 4.2*10^6 eggs and microscope counts estimated 8.88*10^6 eggs

Checked on quality of sperm. 2/3 males had high quality sperm based on appearance and activity.

• 600mL of eggs in 2 tripours
  • Added 6mL of sperm
• Checked on scope for egg:sperm; over 30 egg to sperm
• Added sperm to LRT (5000L tank) after 5 minutes.

The LRT was at 10C which may slow down the fertilization and development process. A sample after ~40 minutes showed some polar body formation.

 

Matt sent over a picture of polar bodies from the LRT later that day:

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.

• • • cfx Report with plate layout, cycling params, etc.
    • table with Cf and melt values, and sex and stage of each sample

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.