Differential Expression of MicroRNA (MiR-27, MiR-145) among Dental Pulp Stem Cells (DPSCs) Following Neurogenic Differentiation Stimuli
Abstract
:1. Introduction
2. Materials and Methods
2.1. Project Approval
2.2. Original Study Approval and Sample Collection
2.3. Original Sample Processing and Storage
2.4. Current Study Protocol
2.5. Growth Factor Administration
2.6. Viability and Proliferation Assays
2.7. RNA Isolation
2.8. cDNA Synthesis
2.9. microRNA Processing
2.10. qPCR Screening
2.11. Statistical Analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primers | Sequence |
---|---|
Positive control primers | |
Forward primer, Beta actin | 5′-GTG GGG TCC TGT GGT GTG-3′ |
Reverse primer, Beta actin | 5′-GAA GGG GAC AGG CAG TGA-3′ |
Forward primer, GAPDH | 5′-ATC TTC CAG GAG CGA GAT CC-3′ |
Reverse primer, GAPDH | 5′-ACC ACT GAC ACG TTG GCA GT-3′ |
MSC primers | |
Forward primer, Nestin | 5′-CGT TGG AAC AGA GGT TGG AG-3′ |
Reverse primer, Nestin | 5′-TCC TGA AAG CTG AGG GAA G-3′ |
Forward primer, NANOG | 5′-GCT GAG ATG CCT CAC ACG GAG-3′ |
Reverse primer, NANOG | 5′-TCT GTT TCT TGA CTG GGA CCT TGT C-3′ |
Forward primer, Oct4 | 5′-TGG AGA AGG AGA AGC TGG AGC AAA A-3′ |
Reverse primer, Oct4 | 5′-GGC AGA TGG TCG TTT GGC TGA ATA-3′ |
Forward primer, Sox2 | 5′-ATG GGC TCT GTG GTC AAG TC-3′ |
Reverse primer, Sox2 | 5′-CCC TCC CAA TTC CCT TGT AT-3′ |
ISCT control primers | |
Forward primer, CD45 | 5′-CAT ATT TAT TTT GTC CTT CTC CCA-3′ |
Reverse primer, CD45 | 5′-GAA AGT TTC CAC GAA CGG-3′ |
Forward primer, CD90 | 5′-ATG AAC CTG GCC ATC AGC A-3′ |
Reverse primer, CD90 | 5′-GTG TGC TCA GGC ACC CC-3′ |
Forward primer, CD105 | 5′-CCA CTA GCC AGG TCT CGA AG-3′ |
Reverse primer, CD105 | 5′-GAT GCA GGA AGA CAC TGC TG-3′ |
Neuronal differentiation primers | |
Forward primer, BIII tubulin | 5′-GGC CAA GGG TCA CTA CAC G-3′ |
Reverse primer, BIII tubulin | 5′-GCA GTC GCA GTT TTC ACA CTC-3′ |
Forward primer, NFM | 5′-GCT CGT CAT TTG CGC GAA TAC-3′ |
Reverse primer, NFM | 5′-TTT CTG TAC GCA GCG ATT TCT AT-3′ |
Forward primer, Pax6 | 5′-TGG GCA GGT ATT ACG AGA CTG-3′ |
Reverse primer, Pax6 | 5′-ACT CCC GCT TAT ACT GGG CTA-3′ |
Forward primer, Sox1 | 5′-CAG TAC AGC CCC ATC TCC AAC-3′ |
Reverse primer, Sox1 | 5′-GCG GGC AAG TAC ATG CTG A-3′ |
Forward primer, Vim | 5′-GAC GCC ATC AAC ACC GAG TT-3′ |
Reverse primer, Vim | 5′-CTT TGT CGT TGG TTA GCT GGT-3′ |
microRNA primers | |
Forward primer, miR-16 | 5′-TAG CAG CAC GTA AAT ATT GGC G-3′ |
Reverse primer, miR-16 | 5′-TGC GTG TCG TGG AGT C-3′ |
Forward primer, miR-27 | 5′-ATA TGA GAA AAG AGC TTC CCT GTG-3′ |
Reverse primer, miR-27 | 5′-CAA GGC CAG AGG AGG TGA G-’3′ |
Forward primer, miR-125 | 5′-GCC CTC CCT GAG ACC TCA A-3′ |
Reverse primer, miR-125 | 5′-GTG CAG GGT CCG AGG T-3′ |
Forward primer, miR-128 | 5′-TCT CCT AAA GAG CCC GAA CA-3′ |
Reverse primer, miR-128 | 5′-TTG CAT TCA TAG CTG CAT CC-3′ |
Forward primer, miR-135 | 5′-CGA TAT GGC TTT TTA TTC CTA -3′ |
Reverse primer, miR-135 | 5′-GAG CAG GGT CCG AGG T -3′ |
Forward primer, miR-140 | 5′-GGG CAG TGG TTT TAC CCT A -3′ |
Reverse primer, miR-140 | 5′-CAG TGC GTG TCG TGG AGT -3′ |
Forward primer, miR-145 | 5′-AGA GAA CTC CAG CTG-3′ |
Reverse primer, miR-145 | 5′-GGC AAC TGT GGG GTG-3′ |
Forward primer, miR-218 | 5′-TCG GGC TTG TGC TTG ATC T-3′ |
Reverse primer, miR-218 | 5′-GTG CAG GGT CCG AGT G-3′ |
Forward primer, miR-410 | 5′-CCG CAC GAT ATA ACA CAG ATG-3′ |
Reverse primer, miR-410 | 5′-GTG CAG GGT CCG AGG TAT TC-3′ |
Rapid Doubling Time (rDT) | Intermediate Doubling Time (iDT) | Slow Doubling Time (sDT) | Viability (Baseline) | |
---|---|---|---|---|
dpsc-7089 | 1.9 days | 62% | ||
dpsc-3882 | 1.8 days | 58% | ||
dpsc-5653 | 1.9 days | 77% | ||
dpsc-9765 | 2.1 days | 69% | ||
dpsc-3924 | 2.2 days | 66% | ||
rDT average | 1.98 days | 66.40% | ||
dpsc-8604 | 5.1 days | 50% | ||
dpsc-8124 | 5.4 days | 53% | ||
dpsc-9894 | 5.2 days | 66% | ||
iDT average | 5.23 days | 56.30% | ||
dpsc-11418 | 10.2 days | 63% | ||
dpsc-17322 | 10.6 days | 56% | ||
dpsc-11836 | 12.1 days | 57% | ||
dpsc-11750 | 11.9 days | 62% | ||
sDT average | 11.2 days | 59.50% |
RNA Concentration | RNA Purity (A260:A280 Ratio) | cDNA Concentration | DNA Purity (A260:A280 Ratio) | |
---|---|---|---|---|
dpsc-7089 | 481 ng/µL | 1.88 | 1541 ng/µL | 1.89 |
dpsc-3882 | 357 ng/µL | 1.67 | 1480 ng/µL | 1.92 |
dpsc-5653 | 443 ng/µL | 1.82 | 1606 ng/µL | 1.82 |
dpsc-9765 | 432 ng/µL | 1.73 | 1587 ng/µL | 1.9 |
dpsc-3924 | 320 ng/µL | 1.82 | 1527 ng/µL | 1.92 |
rDT average | 406.6 ng/µL | 1.78 | 1548.2 ng/µL | 1.89 |
dpsc-8604 | 447 ng/µL | 1.84 | 1589 ng/µL | 1.8 |
dpsc-8124 | 302 ng/µL | 1.66 | 1551 ng/µL | 1.89 |
dpsc-9894 | 577 ng/µL | 1.85 | 1561 ng/µL | 1.81 |
iDT average | 442.0 ng/µL | 1.78 | 1567.0 ng/µL | 1.83 |
dpsc-11418 | 484 ng/µL | 1.77 | 1607 ng/µL | 1.81 |
dpsc-17322 | 519 ng/µL | 1.78 | 1610 ng/µL | 1.89 |
dpsc-11836 | 321 ng/µL | 1.86 | 1489 ng/µL | 1.86 |
dpsc-11750 | 567 ng/µL | 1.85 | 1492 ng/µL | 1.92 |
sDT average | 472.8 ng/µL | 1.81 | 1549.5 ng/µL | 1.87 |
ICST Biomarkers | MSC Biomarkers | Neuronal Biomarkers (Post-Treated) | microRNA Expression | |
---|---|---|---|---|
rDT dpsc-7089 dpsc-2883 dpsc-5653 dpsc-9765 dpsc-3924 | CD90, CD105 (all rDT) CD45 (no rDT) | NANOG Nestin Oct4 Sox2 (all rDT) | BIII tubulin, NFM (all rDT) Pax, Sox1, Vim (all rDT but dpsc-3882) | miR-27 (all rDT but dpsc-5653) miR-145 (dpsc-7089, dpsc-3882, dpsc-9765) miR-125, miR-128 miR-135 miR-140 miR-218 miR-410 (no rDT) |
iDT dpsc-8604 dpsc-8124 dpsc-9894 | CD90 (all iDT) CD105 (all iDT) CD45 (no iDT) | NANOG Nestin Oct4 Sox2 (all iDT) | BIII tubulin NFM Pax Sox1 Vim (no iDT) | miR-27 miR-125, miR-128 miR-135 miR-140 miR-145 miR-218 miR-410 (no iDT) |
sDT dpsc-11418 dpsc-17322 dpsc-11836 dpsc-11750 | CD90 (all sDT) CD105 (all sDT) CD45 (no sDT) | NANOG (dpsc-17322) Nestin (all sDT) Sox2, Oct4 (no sDT) | BIII tubulin NFM Pax Sox1 Vim (no sDT) | miR-27 (all sDT) miR-145 (all sDT) miR-125, miR-128 miR-135 miR-140 miR-218 miR-410 (no sDT) |
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Bassett, C.; Triplett, H.; Lott, K.; Howard, K.M.; Kingsley, K. Differential Expression of MicroRNA (MiR-27, MiR-145) among Dental Pulp Stem Cells (DPSCs) Following Neurogenic Differentiation Stimuli. Biomedicines 2023, 11, 3003. https://doi.org/10.3390/biomedicines11113003
Bassett C, Triplett H, Lott K, Howard KM, Kingsley K. Differential Expression of MicroRNA (MiR-27, MiR-145) among Dental Pulp Stem Cells (DPSCs) Following Neurogenic Differentiation Stimuli. Biomedicines. 2023; 11(11):3003. https://doi.org/10.3390/biomedicines11113003
Chicago/Turabian StyleBassett, Charlton, Hunter Triplett, Keegan Lott, Katherine M. Howard, and Karl Kingsley. 2023. "Differential Expression of MicroRNA (MiR-27, MiR-145) among Dental Pulp Stem Cells (DPSCs) Following Neurogenic Differentiation Stimuli" Biomedicines 11, no. 11: 3003. https://doi.org/10.3390/biomedicines11113003