Kaitlyn’s notebook: sex/stage plots on primers

Primers by sex & stage:

sex-stage-cqmean

Female Cq.Means by Stage:

female-cqmean

Male Cq.Means by Stage:

male-cqmean

 

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

All primer pairs by sex and stage:

primers-Cqmean

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.

stage-Cqmean

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.


Primers by Stage:

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Primers by Sex:

sex-Cqmean

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

sex-peakheight-zeros

A value of 0 was plotted for values not reported.

sex-melttemp-zeros

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.


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

meltcurvemeltpeakqpcr2

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

  1. Developed primers for biomarkers

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.

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

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.


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.

20200205_102703


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

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). 20200127_134701.jpg

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!