Primers by sex & stage:
Female Cq.Means by Stage:
Male Cq.Means by Stage:
Previous qPCR test on pooled samples determined these 4 primers would be run on known samples. Ran qPCR as previously described.
Two runs had to be done to be able to include all samples.
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:
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.
I made cDNA from previously isolated RNA. I included a pooled sample that I used to test the primers I recently designed via qPCR.
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.
Samples are stored in the -20C fridge in 209 in 20190107 Kaitlyn’s Box.
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.
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!
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).
1.Identified biomarkers for reproductive development/sex
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.
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 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 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.
|23||F||2||low (151.89 RFU)|
|37||F||6||high (4893.75 RFU)|
|43A||M||4||high (3520.34 RFU)|
|54||F||3||high (3152.78 RFU)|
|55F||F||7||high (3544.7 RFU)|
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.
I also picked up the new primers from Biochem stores which are on the benchtop in 209.
Isolated RNA from the following hemolymph pellet samples:
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:
Samples 58 and 61 were clear (left) in contrast to the cloudy appearance of the other samples (right).
Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch) according to the manufacturer’s protocol for liquids/cells in RNAlater.
RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.
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!
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.
pyfaidx– splits multi-FastA into individual FastA files
Primer3– performs primer design
EMBOSS(installation issue linked) – checks primer specificity against entire transcriptome
Primer3(issue with code)
Shelly, Brent and I helped Matt with a strip spawn yesterday!
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.
Checked on quality of sperm. 2/3 males had high quality sperm based on appearance and activity.
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:
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:
Here is my qPCR master mix calculations. Sam previously ran a qPCR using the above samples. All qPCR reactions were run in duplicate.
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.