qPCR for Nickel exposure on Botryllus schlosseri

Relative standard curves to determine primer effenciencies.

nickel chloride
qPCR
primers
Author

Celeste Valdivia

Published

March 1, 2024

Objective

Analysis of gene expression with quantitative Polymerase Chain Reaction (qPCR) will be a component of the in vivo nickel exposure work conducted in autumn 2023. Four experiments were completed using a high and low dose of nickel chloride (5 and 45 mg L-) and we are aiming to evaluate the impact of nickel toxicity on gene expression relating to oxidative stress, senescence, stemness, and apoptosis.

Background

Refer to pilot exposure post for more details regarding procedures and experimental design.

Experiment was repeated three more times after pilot exposure to increase the number of replicates per treatment.

Final experimental set up

Nickel Concentration (mg/L) Exposure Length (hrs) Number of Replicates
0 24 14
5 24 13
45 24 12

Protocol leading to cDNA synthesis

  1. RNA extracted using Qiagen RNeasy Mini Kit with 10% β-mercaptoethanol (β-ME) in RLT buffer and QiaShredder column step supplemented to protocol.

  2. Quantify RNA with NanoDrop.

  3. gDNA elimination with routine DNAse treatment with Invitrogen TURBO DNA-free Kit (Cat. No. AM1907).

gDNA Elimination Component per 1 rxn (uL)
10x DNase Buffer 2
Turbo Enzyme 1
RNA sample 20
Total 23

2uL of DNase Inactivation Reagent used towards end of protocol.

  1. Requantification of gDNA eliminated RNA with Nano Drop:
Date Sample ID RNA Concentration (ng/uL) A260/A280 260(10mm)
09/30/2023 9302023 0-R1 pilot 19.2 1.55 0.48
09/30/2023 9302023 0-R2 pilot 113.2 1.85 2.83
09/30/2023 9302023 0-R3 pilot 152.4 1.87 3.809
09/30/2023 9302023 0-R4 pilot 67.1 1.77 1.676
09/30/2023 9302023 5-R1 pilot 22.4 1.66 0.56
09/30/2023 9302023 5-R2 pilot 30.6 1.68 0.764
09/30/2023 9302023 5-R3 pilot 29 1.67 0.726
09/30/2023 9302023 5-R4 pilot 45.1 1.77 0.128
09/30/2023 9302023 45-R2 pilot 87.6 1.82 2.14
09/30/2023 9302023 45-R3 pilot 13.8 1.47
09/30/2023 9302023 45-R4 pilot 84.8 1.83 2.12
10/25/2023 10252023 0-R1 E1 12.1 1.55 0.305
10/25/2023 10252023 0-R2 E1 45.9 1.83 1.148
10/25/2023 10252023 0-R3 E1 78.4 1.85 1.959
10/25/2023 10252023 0-R4 E1 38.5 1.8 0.962
10/25/2023 10252023 5-R1 E1 83.3 1.82 2.082
10/25/2023 10252023 5-R3 E1 27.1 1.69 0.676
10/25/2023 10252023 5-R4 E1 69.5 1.84 1.737
10/25/2023 10252023 45-R1 E1 50 1.78 1.249
10/25/2023 10252023 45-R2 E1 57.3 1.84 1.433
10/25/2023 10252023 45-R3 E1 43.5 1.73 1.087
10/25/2023 10252023 45-R4 E1 60.1 1.83 1.505
11/15/2023 11152023 0-R1 E2 86.5 1.86 2.163
11/15/2023 11152023 0-R2 E2 54.7 1.66 1.367
11/15/2023 11152023 0-R3 E2 37.4 1.72 0.935
11/15/2023 11152023 5-R1 E2 58.4 1.66 1.459
11/15/2023 11152023 5-R2 E2 69.7 1.69 1.743
11/15/2023 11152023 5-R3 E2 33.7 1.51 0.843
11/15/2023 11152023 45-R2 E2 34.5 1.44 0.863
11/15/2023 11152023 45-R3 E2 16.2 1.5 0.405
11/29/2023 11292023 0-R1 E3 72.4 1.78 1.809
11/29/2023 11292023 0-R2 E3 60.3 1.78 1.507
11/29/2023 11292023 0-R3 E3 21.4 1.61 0.534
11/29/2023 11292023 5-R1 E3 12.7 1.43 0.318
11/29/2023 11292023 5-R2 E3 15.5 1.57 0.387
11/29/2023 11292023 5-R3 E3 64.4 1.76 1.609
11/29/2023 11292023 45-R1 E3 80.6 1.82 2.016
11/29/2023 11292023 45-R2 E3 49.3 1.77 1.231
11/29/2023 11292023 45-R3 E3 64.3 1.79 1.607
Warning

RNA quality drop difference of about 0.2 after gDNA elimination.

  1. cDNA synthesis with SuperScript IV VILO Kit.
cDNA synthesis Component per 1 rxn (uL)
SuperScript IV VILO Master Mix 2
Nuclease-free water variable
RNA sample variable
Total 10
Note

Final mass of 96.8 ng of RNA in reaction mix for each sample (thus variable water and RNA volumes)

cDNA Pool

Note

It is not recommended to NanoDrop cDNA as readings below may be inaccurate due to residual nucleotide and etc. from the synthesis reaction. Assume the reaction is a 1:1 RNA:cDNA.

cDNA pool spectrometer readings and dilutions:

Date Made Sample cDNA ng/uL A260/A280
12/18/2023 Original Pool 2231.2 1.73
1/14/2024 10-2 223.12 1.73
1/14/2024 10-3 22.312 1.73
1/14/2024 10-4 2.312 1.73
1/14/2024 10-5 0.2312 1.73
1/14/2024 10-6 0.02312 1.73

Primers

Primers for Botryllus schlosseri designed as described in Tunicate Primers notebook entry.

Number Gene Forward Primer (5’-3’) Reverse Primer (5’-3’) NCBI Accession Number (If complete CDS) Botryllus Contig Sequence (if no complete CDS was found) Amplicon Length Source
1 BAX CATTGACCTTGCGTGTTATTGG AAGATGTCCTTGGTGGGTTG KU948200.1 None 97 in house
2 POU3 CAGTAGGTCTTCGCATCCATC CGTTTGCATCGTTGGTGTAAG KU948203.1 None 80 in house
3 PolB GGCTTCACAATCAATGAGTATTCC AGCAATCTTTCCGCATTCATTC None botctg092056 NA in house
4 TNF GTTTCAGTCAACACCTTGTGTC AATCTCGACCGCTGACTTATAC None botctg065850 131 in house
5 SOXB1 GGGAATGTGTTCATCCTCTGG TGAATCCAGGAGTTACTCAAGTTC KU948203.1 None 116 in house
6 MYC GAAGTCCTGGCTAAGATGCTAC AGCGTCAAGAAGGAAGTACG KU948203.1 None 119 in house
7 AIF1 GGATTCTCTCACCGATGGAATAG CCTGACTTGGAGCCTTTATGG KU948202.1 None 102 in house
8 SOD1 CAATCCATATAACAAGGTTCATGGC TGATATCCACACAGGCACAAC EE743567.1 None 107 in house
9 PARP1 AATTTGGCACCGCTCATTTC TGCAAGACCTTCAGCTTCTC KU948201.1 None 94 in house
10 SIRT6 ATTAGTACCGCGTCTGGAATAC GGATGGAATCGCTTCTTCAAATC None botctg109566 114 in house
11 IAP7 GGTCTCTTCACGGACATCTTC GCTCAGTATGTTGTGCTTGTTC KU948203.1 None 111 in house
12 HSP70 CGAGAAGTTGAAGGACAAGATCTCCG TCTCTGCAGTCTGGTTGTTGTCGAGC NA NA NA doi:10.1016/j.ydbio.2017.10.023
13 Mortalin TTCATGATACCGAAACCAAGATGGACG AATTTCCTCCTTGATGGCATCCACC KY212120.1 None 79 doi:10.1016/j.ydbio.2017.10.023
14 ACTb GTAGGTAGTCTCGTGAATTC CACGCCATCTTGCGTCTGGA NA NA 321 doi:10.1016/j.dci.2006.12.009
15 18s GGCAGCCTCCGGGAAACCAAAGTC CTGGTGGTGCCCTTCCGTCAATTC NA NA NA doi:10.1016/j.ydbio.2017.10.023

End-Point PCR

Figure 1: EP-PCR conducted to confirm correct size of amplicon products. In this PCR reaction run, primer sets were at a concentration of 5 uM. Note this is different than what was used for the 1:10 dilution series relative standard curves. Also where it says 40s, it should say 18s.

EP-PCR Reaction Info

EP-PCR Component per 1 rxn (uL)
PCR Master Mix (ThermoFisher) 12.5
Nuclease-free water 9.5
Forward Primer (5 uM) 1
Reverse Primer (5 uM) 1
1:3 dilution of pooled (per treatement) cDNA 1
Total 25
Note

Final concentration of 200 nM of each primer per 1 rxn.

Gel Info

With 1x TAE buffer on 1% agarose gel:

Component per 1 rxn (uL)
Nuclease-free water 5
cDNA (after EP-PCR) 5
Loading Dye 2
Total 12

Relative Standard Curves

To view data and results directly from plate reader view:

01-15-2024 Plate 1

01-29-2024 Plate 1

01-29-2024 Plate 2

qPCR Reaction Information

Component per 1 rxn (uL)
Applied Biosystems™ PowerSYBR™ Green PCR Master Mix 5
Nuclease-free water 3.5
Forward Primer (2.5 uM) 0.25
Reverse Primer (2.5 uM) 0.25
cDNA 1
Total 10
Note

Final concentration of 62.5 nM of each primer per 1 rxn (suggested to be within 50-300 nM for this protocol).

Cycle Information

Stage Step Temperature (C) Duration (sec) Cycles
Hold Hold 50 120 1
Hold Hold 95 600 1
PCR Denature 95 15 40
PCR Anneal/Extend 60 60 40
Melt Curve Denature 95 15 1
Melt Curve Anneal/Extend 60 60 1
Melt Curve Dissociation 95 1 1
Figure 2: Cycle information for qPCR.

NTC results

Target Name Task CT
BActin NTC Undetermined
BAX NTC 32.548
SOD1 NTC 26.921
SIRT6 NTC Undetermined
IAP7 NTC 32.048
HSP70 NTC Undetermined
Mortalin NTC 32.660
18s NTC 32.623
POU3 NTC 33.793
PolB NTC Undetermined
TNF NTC Undetermined
MYC NTC 32.153
AIF1 NTC 27.873
PARP1 NTC Undetermined

Good

Beta actin

The two primer sets with acceptable efficiency values in my qPCR are my housekeeping (HK) genes. I suspect given they are constitutively and highly expressed, issues with inhibitors in the reaction mix are not as much of an issue relative to my genes of interest (GOI).

Figure 3: Efficiency for beta actin is 94%

18s

Figure 4: Efficiency value for 18s = 84%.

Questionable

BAX

Figure 5: Efficiency for BAX is 350%

AIF1

Figure 6: Efficiency for AIF1 is 148,350.875

Mortalin

Figure 7: Efficiency for Mortalin is 174.206

SIRT6

Figure 8: Efficiency for SIRT6 is 176.364.

SOD1

Figure 9: Efficiency for SOD1 is 2,189,061.750%

POU3

Figure 10: Efficiency for POU3 is 673%.

SOXB1

Figure 11: Efficiency for SOXB1 is 364.444

MYC

Figure 12: Efficiency for MYC is 405.222

IAP7

Figure 13: Efficiency for IAP7 is 299.488

Trash

PolB

Figure 14: Basically no amplification, E = 26.604.

TNF

Figure 15: Basically no amplification, E = 918.266.

HSP70

Figure 16: Basically no amp, E = 52.113.

PARP1

Figure 17: Late amp, E = 117.058%

Discussion

cDNA has low purity?

According to online sources DNA purity (260/280) should fall above 1.8. Currently the cDNA is at 1.7. RNA should fall above 2.0 (260/280). The RNA after gDNA elimination fell below that at an average of 1.71 (SD ± 0.12).

When running all the primer sets at a 1:5 dilution series, higher concentrations of cDNA resulted in a ‘horseshoe’ shape (indicative of failure to fluoresce) and most diluted sample (1:625) had lower Ct values than higher concentrations (1:125). Upon running a 1:10 dilution series, this issue is no longer as apparent. However, online sources mention that an amplification efficiency above 100% (which is what we see for nearly all the primers) or a really flat slope on the standard curve is indicative of polymerase inhibition.

Figure 18: 18s primer amplification plot at a 1:5 dilution series. Downward parabola shapes are higher concentrations of the cDNA pools.
Figure 19: Notice that in some of the wells, after the qPCR reaction, there appears to be an unknown red precipitate at the bottom of the well. Appears as if there is more “red” in top right quadrant loaded with SOD1 primer. Other plates not fully assessed for this phenomenon.

Common contaminants:

  • hemoglobin

  • polysaccharides

  • proteinase K

  • ethanol (during the RNA isolation)

However, the purity of my RNA prior to gDNA elimination was on average 1.89 (SD ± 0.11) so likely reagents fromt the gDNA elimination step were carried over into my samples.

Primers are not optimally designed?

Only workable primer sets will be those with a singular peak and appear to fail to have an acceptable efficiency curve due to high standard deviation across replicates.

The following primers currently in stock will be utilized moving forward:

Potentially Workable Primers of the Bunch

Number Gene Forward Primer (5’-3’) Reverse Primer (5’-3’) Source
1 BAX CATTGACCTTGCGTGTTATTGG AAGATGTCCTTGGTGGGTTG in house
7 AIF1 GGATTCTCTCACCGATGGAATAG CCTGACTTGGAGCCTTTATGG in house
10 SIRT6 ATTAGTACCGCGTCTGGAATAC GGATGGAATCGCTTCTTCAAATC in house
13 Mortalin TTCATGATACCGAAACCAAGATGGACG AATTTCCTCCTTGATGGCATCCACC doi:10.1016/j.ydbio.2017.10.023
14 ACTb GTAGGTAGTCTCGTGAATTC CACGCCATCTTGCGTCTGGA doi:10.1016/j.dci.2006.12.009
15 18s GGCAGCCTCCGGGAAACCAAAGTC CTGGTGGTGCCCTTCCGTCAATTC doi:10.1016/j.ydbio.2017.10.023

Work arounds

Include Clean Up Step

Issue may lie completely with the low quality of the RNA at the moment. Using different primers potentially wouldn’t improve outcome since the puritiy falls bellow the acceptable threshold.

Alternative Primers

Further literature search regarding previous work conducted with B. schlosseri and qPCR resulted in finding the following paper:

Citation

Rosner A, Kravchenko O, Rinkevich B. IAP genes partake weighty roles in the astogeny and whole body regeneration in the colonial urochordate Botryllus schlosseri. Developmental Biology. 2019 Apr;448(2):320–41. doi.org/10.1016/j.ydbio.2018.10.015

This paper utilizes several primer sets targeting genes in relation to apoptosis. The following primer sets are pulled from this paper:

Proposed Primers

Gene* or Accession** number Gene name Forward primer Reverse primer Source
g5785 IAP6 CTGGTATTGTAACGGCGGCCTGCAA GGTGAACGAAATGCTGGCCTTTGGA doi.org/10.1016/j.ydbio.2018.10.015
g7904 IAP8 ATTTACCTCGGACGTTAGCGCAGGAC CCCGGAACAAGAGAAGCATTGAACTC doi.org/10.1016/j.ydbio.2018.10.015
g7149 IAP9 GGATGCGGGATATGCGGAGGATAG AGCTTCCGCCGACTGATAGCATG doi.org/10.1016/j.ydbio.2018.10.015
g27485 IAP14 GCGTGACCGTGTGAAATGCTGG CCACTTTGCATGCTCGACCCAC doi.org/10.1016/j.ydbio.2018.10.015
g44848 IAP15 ATGGAAGCCACCGACGAGATGAG TCGATAACGTTCTGTTGGCAGCTCTTG doi.org/10.1016/j.ydbio.2018.10.015
g56072 IAP18 TAAGCACATTCATGCGGTGGTCG GCCCAAATAGAACAATCCGCAGTCTG doi.org/10.1016/j.ydbio.2018.10.015
g29315 IAP21 CGTGAGCCTCTACTGCGCACTTCTC GCTTGAGTAGAAGTTGGCGCAGAGC doi.org/10.1016/j.ydbio.2018.10.015
KU948200.1** Bax ATCTAGGCCAACCCGACCGAGAACC CAACAGCTCCCTGGACCAAAGACG doi.org/10.1016/j.ydbio.2018.10.015
KU948202.1** AIF1 GGTGAGCAAGGGCATAGAAC TGAACGACTCCGTGTTTGG doi.org/10.1016/j.ydbio.2018.10.015
g8767 caspase 2 TGGTGAAGGTCCTGGCGAAGAAC GCCTTTAACGACGCTGTTGACGG doi.org/10.1016/j.ydbio.2018.10.015
g11647 caspase7.1 GCACAAATTCCGTGGTTGCTTCC TTCTCTTGTCTCTGTGCGTCCCGTTTC doi.org/10.1016/j.ydbio.2018.10.015
g3334 caspase9 GAAACCGATCTTACCGCCTACAACATG ACGCATATATCAGATCGTCCTTGTTGC doi.org/10.1016/j.ydbio.2018.10.015
g49756 AKT1 CTTGAAGACAATGATTACGGCAGGTCC AAGCGTCCGCACATCATCTCGTAC doi.org/10.1016/j.ydbio.2018.10.015
g1445 PIK3CA CTGATAAGGAAAGTCCACGGCAACGAC CTGACCGGTTTGACGTGGAGTAATTG doi.org/10.1016/j.ydbio.2018.10.015
g42386 GSK3b2 GTTTCCGCAAATCAAGGCTCATCC TGCTACATAAACCGATGGCGTCCTG doi.org/10.1016/j.ydbio.2018.10.015
g12744 cdc25 GAAATGTTCCCTGCCCACTGTGAC CGATGCCGTGTTCTTTCTTCAGAGC doi.org/10.1016/j.ydbio.2018.10.015
g41847 myc GCAGATCAGGGCCGCTCACAATGTC TGTCGCGCAGCGTCAAGAAGGAAG doi.org/10.1016/j.ydbio.2018.10.015
g61081 sox2 ACGCAGTTAATCATCATCCATCTCACC AACCGGGATGTGGGCTACTTGC doi.org/10.1016/j.ydbio.2018.10.015
g68340 hsp90α 3_1 AAGATCATTTAGCCGTCAAGCACTTCTC GGGCACGCTTTGGAACGAAGAG doi.org/10.1016/j.ydbio.2018.10.015
g67511 hsp90β 2_4 AAGGCCCAGGCTCTTCGTGACTCTTC GAGTGTTCGGGATTGATCTCCAAGTG doi.org/10.1016/j.ydbio.2018.10.015
Note

*Gene number assigned by the Botrullus Genome Project (Voskoboynik et al., 2013).

**Accession number assigned by the NCBI GenBank.

Of the suite of IAPs reduced from the ones used in the referenced paper to capture those that were most interesting under oxidative stress at stage A and across blastogenic stages.

In our study we did not target a specific stage for oxidative stress (nickel) exposures so I believe it would be interesting to capture IAPs impacted by both factors.