Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels
Abstract
:1. Introduction
2. Results
2.1. High-Quality RNA Can Be Extracted from BM-MSCs within MeHA Using Double Phase Separation
2.2. The Type of Reverse Transcriptase Affects the ∆Cq Values During Real Time PCR
2.3. The Culture Type (2D vs. 3D) Influences the Performance of a Reference Gene
2.4. The Stability of a Reference Gene Differs between 2D and 3D Culture
2.5. The Reference Gene of Choice Has an Impact on the Target Gene Expression
2.6. Osteogenic Differentiation of hBM-MSCs within MeHA Hydrogels
3. Discussion
4. Materials and Methods
4.1. Methacrylated Hyaluronic Acid Polymer Synthesis
4.2. Cell Isolation and Culture
4.3. Cell Laden Methacrylated Hyaluronic Acid Hydrogel Preparation
4.4. Osteogenic Differentiation of 2D and 3D Culture
4.5. RNA Isolation from 2D and 3D Culture
4.6. RNA Assessment
4.7. qRT-PCR
4.8. Reference Gene Validation
4.8.1. geNorm
4.8.2. NormFinder
4.8.3. ΔCq Method
4.9. Statistics
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ALPL | Alkaline phosphatase |
BCP | 1-Bromo-3-chloropropane |
BM-MSCs | Bone marrow-derived mesenchymal stromal cells |
COL1A1 | Collagen type 1 |
COL10A1 | Collagen type 10 |
Cq | Cycle quantification |
∆Cq | Delta cycle quantification |
EtOH | Ethyl alcohol |
FBS | Fœtal bovine serum |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
GUSB | Beta-glucuronidase |
HA | Hyaluronic acid |
IBSP | Bone sialoprotein |
IPA | Isopropanol alcohol |
MeHA | Methacrylated hyaluronic acid |
OAZ1 | Ornithine Decarboxylase Antizyme 1 |
OC | Osteocontrol |
OG | Osteogenic |
PCR | Polymerase chain reaction |
PBS | Phosphate buffered saline |
PPARγ | Peroxisome proliferator-activated receptor gamma |
PPIA | Peptidylprolyl Isomerase A |
RUNX2 | Runt-related transcription factor 2 |
RT-qPCR | Reverse transcription quantitative polymerase chain reaction |
RIN | RNA integrity number |
RPLP0 | Ribosomal Protein Lateral Stalk Subunit P0 |
SD | Standard deviation |
SOX9 | Sex determining region Y box 9 |
TBP | TATA box binding protein |
YWHAZ | Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta |
18S | 18S ribosomal RNA |
Appendix A
2D | 3D | |||||
---|---|---|---|---|---|---|
Comparison | ∆Cq Mean | SD | Mean SD | ∆Cq Mean | SD | Mean SD |
18S vs. GAPDH | 14.19 | 0.49 | 13.61 | 0.71 | ||
18S vs. GUSB | 20.08 | 0.30 | 18.33 | 0.70 | ||
18S vs. OAZ1 | 16.43 | 0.39 | 16.42 | 0.53 | ||
18S vs. PPIA | 15.16 | 0.56 | 15.30 | 0.43 | ||
18S vs. RPLP0 | 11.83 | 0.38 | 13.19 | 0.63 | ||
18S vs. TBP | 21.44 | 0.53 | 22.33 | 0.85 | ||
18S vs. YWHAZ | 22.99 | 0.70 | 0.48 | 22.97 | 0.89 | 0.92 |
RPLP0 vs. GAPDH | 2.36 | 0.26 | 0.92 | 0.56 | ||
RPLP0 vs. GUSB | 8.25 | 0.24 | 5.15 | 0.66 | ||
RPLP0 vs. OAZ1 | 4.61 | 0.34 | 3.24 | 0.36 | ||
RPLP0 vs. PPIA | 3.33 | 0.37 | 2.11 | 0.44 | ||
RPLP0 vs. TBP | 9.61 | 0.33 | 9.14 | 0.69 | ||
RPLP0 vs. YWHAZ | 11.16 | 0.45 | 9.79 | 0.98 | ||
RPLP0 vs. 18S | 11.83 | 0.38 | 0.34 | 13.19 | 0.63 | 0.62 |
GAPDH vs. GUSB | 5.89 | 0.38 | 4.73 | 1.33 | ||
GAPDH vs. OAZ1 | 2.25 | 0.29 | 2.82 | 1.03 | ||
GAPDH vs. PPIA | 0.97 | 0.24 | 1.69 | 0.92 | ||
GAPDH vs. RPLP0 | 2.36 | 0.26 | 0.92 | 0.56 | ||
GAPDH vs. TBP | 7.25 | 0.27 | 8.72 | 1.44 | ||
GAPDH vs. YWHAZ | 8.80 | 0.52 | 9.37 | 1.48 | ||
GAPDH vs. 18S | 14.19 | 0.49 | 0.35 | 13.61 | 0.71 | 1.76 |
PPIA vs. GAPDH | 0.97 | 0.24 | 1.69 | 0.92 | ||
PPIA vs. GUSB | 4.92 | 0.47 | 3.03 | 0.50 | ||
PPIA vs. OAZ1 | 1.27 | 0.32 | 1.12 | 0.21 | ||
PPIA vs. RPLP0 | 3.33 | 0.37 | 2.11 | 0.44 | ||
PPIA vs. TBP | 6.28 | 0.26 | 7.03 | 0.64 | ||
PPIA vs. YWHAZ | 7.83 | 0.55 | 7.68 | 0.75 | ||
PPIA vs. 18S | 15.16 | 0.56 | 0.40 | 15.30 | 0.43 | 0.56 |
OAZ1 vs. GAPDH | 2.25 | 0.29 | 2.82 | 1.03 | ||
OAZ1 vs. GUSB | 3.65 | 0.44 | 1.91 | 0.42 | ||
OAZ1 vs. PPIA | 1.27 | 0.32 | 1.12 | 0.21 | ||
OAZ1 vs. RPLP0 | 4.61 | 0.34 | 3.24 | 0.36 | ||
OAZ1 vs. TBP | 5.01 | 0.36 | 5.90 | 0.52 | ||
OAZ1 vs. YWHAZ | 6.56 | 0.60 | 6.55 | 0.75 | ||
OAZ1 vs. 18S | 16.43 | 0.39 | 0.39 | 16.42 | 0.53 | 0.55 |
GUSB vs. GAPDH | 5.89 | 0.38 | 4.73 | 1.33 | ||
GUSB vs. OAZ1 | 3.65 | 0.44 | 1.91 | 0.42 | ||
GUSB vs. PPIA | 4.92 | 0.47 | 3.03 | 0.50 | ||
GUSB vs. RPLP0 | 8.25 | 0.24 | 5.15 | 0.66 | ||
GUSB vs. TBP | 1.36 | 0.43 | 3.99 | 0.26 | ||
GUSB vs. YWHAZ | 2.91 | 0.61 | 4.64 | 0.42 | ||
GUSB vs. 18S | 20.08 | 0.30 | 0.41 | 18.33 | 0.70 | 0.61 |
TBP vs. GAPDH | 7.25 | 0.27 | 8.72 | 1.44 | ||
TBP vs. GUSB | 1.36 | 0.43 | 3.99 | 0.26 | ||
TBP vs. OAZ1 | 5.01 | 0.36 | 5.90 | 0.52 | ||
TBP vs. PPIA | 6.28 | 0.26 | 7.03 | 0.64 | ||
TBP vs. RPLP0 | 9.61 | 0.33 | 9.14 | 0.69 | ||
TBP vs. YWHAZ | 1.55 | 0.57 | 0.65 | 0.47 | ||
TBP vs. 18S | 21.44 | 0.53 | 0.39 | 22.33 | 0.85 | 0.70 |
YWHAZ vs. GAPDH | 8.80 | 0.52 | 9.37 | 1.48 | ||
YWHAZ vs. GUSB | 2.91 | 0.61 | 4.64 | 0.42 | ||
YWHAZ vs. OAZ1 | 6.56 | 0.60 | 6.55 | 0.75 | ||
YWHAZ vs. PPIA | 7.83 | 0.55 | 7.68 | 0.75 | ||
YWHAZ vs. RPLP0 | 11.16 | 0.45 | 9.79 | 0.98 | ||
YWHAZ vs. TBP | 1.55 | 0.57 | 0.65 | 0.47 | ||
YWHAZ vs. 18S | 22.99 | 0.70 | 0.57 | 22.97 | 0.89 | 0.82 |
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Donor | Time Point (Day) | Concentration (ng/µL) | A260/280 | A260/230 | Total RNA (ng) |
---|---|---|---|---|---|
1 | 0 | 888.20 | 1.99 | 1.79 | 17,764 |
28 | 278.85 | 1.97 | 1.09 | 5577 | |
2 | 0 | 825.61 | 1.99 | 1.75 | 16,512 |
28 | 231.58 | 1.97 | 0.91 | 4632 | |
3 | 0 | 769.96 | 1.99 | 1.76 | 15,399 |
28 | 269.77 | 1.97 | 1.25 | 5395 | |
4 | 0 | 626.97 | 1.86 | 1.80 | 12,539 |
28 | 222.26 | 1.80 | 1.16 | 4445 |
2D | 3D | |||||||
---|---|---|---|---|---|---|---|---|
Gene Symbol | Cq Day 0 1 | Cq Day 28 1 | ∆Cq | CV 2 | Cq Day 0 1 | Cq Day 28 1 | ∆Cq | CV 2 |
18S | 7.43 ± 0.67 | 7.42 ± 0.24 | −0.02 | 6.71 | 6.81 ± 0.40 | 7.25 ± 0.25 | 0.44 | 5.66 |
RPLP0 | 19.16 ± 0.39 | 19.40 ± 0.40 | 0.24 | 2.07 | 20.04 ± 0.40 | 20.39 ± 0.51 | 0.35 | 2.36 |
GAPDH | 21.45 ± 0.39 | 21.81 ± 0.22 | 0.36 | 1.65 | 19.85 ± 0.62 | 21.41 ± 0.36 | 1.56 | 4.57 |
PPIA | 22.32 ± 0.23 | 22.92 ± 0.15 | 0.60 | 1.60 | 22.26 ± 0.20 | 22.39± 0.28 | 0.13 | 1.09 |
OAZ1 | 23.69 ± 0.54 | 24.05 ± 0.10 | 0.36 | 1.76 | 23.42 ± 0.20 | 23.48 ± 0.34 | 0.06 | 1.15 |
GUSB | 27.48 ± 0.54 | 27.54 ± 0.36 | 0.06 | 1.64 | 25.69 ± 0.26 | 25.03 ± 0.35 | −0.66 | 1.78 |
TBP | 28.53 ± 0.40 | 29.26 ± 0.26 | 0.72 | 1.73 | 29.74 ± 0.38 | 28.96 ± 0.46 | −0.78 | 1.96 |
YWHAZ | 30.34 ± 0.35 | 30.58 ± 0.72 | 0.25 | 1.83 | 30.58 ± 0.34 | 29.42 ± 0.36 | −1.16 | 2.29 |
2D | ||||
Gene Symbol | geNorm | NormFinder | ΔCq Method | Comprehensive Ranking 1 |
RPLP0 | 3 | 1 | 1 | 1 |
GAPDH | 1 | 2 | 2 | 2 |
TBP | 2 | 4 | 3 | 4 |
GUSB | 5 | 6 | 6 | 6 |
OAZ1 | 4 | 3 | 4 | 5 |
PPIA | 1 | 5 | 5 | 3 |
18S | 6 | 7 | 7 | 7 |
YWHAZ | 7 | 8 | 8 | 8 |
3D | ||||
Gene Symbol | geNorm | NormFinder | ΔCq Method | Comprehensive Ranking 1 |
OAZ1 | 1 | 2 | 1 | 2 |
PPIA | 1 | 1 | 2 | 1 |
GUSB | 2 | 3 | 3 | 3 |
TBP | 3 | 6 | 5 | 5 |
RPLP0 | 4 | 5 | 4 | 4 |
18S | 5 | 4 | 7 | 6 |
YWHAZ | 6 | 7 | 6 | 7 |
GAPDH | 7 | 8 | 8 | 8 |
Gene Symbol | Assay ID 1 | Forward/Reverse/Probe |
---|---|---|
18S | Hs99999901_s1 | |
GAPDH | Hs99999905_m1 | |
GUSB | Hs99999908_m1 | |
OAZ1 | Hs00427923_m1 | |
PPIA | Hs99999904_m1 | |
RPLP0 | 5′-TGG GCA AGA ACA CCA TGA TG-3′ 5′-CGG ATA TGA GGC AGC AGT TTC-3′ 5′-AGG GCA CCT GGA AAA CAA CCC AGC-3′ | |
TBP | Hs00427620_m1 | |
YWHAZ | Hs00237047_m1 |
Gene Symbol | Assay ID 1 | Forward/Reverse/Probe |
---|---|---|
ALPL | Hs00758162_m1 | |
COL1A1 | 5′-CCC TGG AAA GAA TGG AGA TGA T-3′ 5′-ACT GAA ACC TCT GTG TCC CTT CA-3′ 5′-CGG GCA ATC CTC GAG CAC CC-3′ | |
COL10A1 | 5′-ACG CTG AAC GAT ACC AAA TG-3′ 5′-TGC TAT ACC TTT ACT CTT TAT GGT GTA-3′ 5′-ACT ACC CAA CAC CAA GAC ACA GTT CTT CAT TCC-3′ | |
IBSP | Hs0017320_m1 | |
PPARγ | Hs00234592_m1 | |
RUNX2 | 5′-AGC AAG GTT CAA CGA TCT GAG AT-3′ 5′-TTT GTG AAG ACG GTT ATG GTC AA-3′ 5′-AGG GCA CCT GGA AAA CAA CCC AGC-3′ | |
SOX9 | Hs00165814_m1 |
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Hasler, J.; Hatt, L.P.; Stoddart, M.J.; Armiento, A.R. Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels. Int. J. Mol. Sci. 2020, 21, 9195. https://doi.org/10.3390/ijms21239195
Hasler J, Hatt LP, Stoddart MJ, Armiento AR. Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels. International Journal of Molecular Sciences. 2020; 21(23):9195. https://doi.org/10.3390/ijms21239195
Chicago/Turabian StyleHasler, Johannes, Luan Phelipe Hatt, Martin James Stoddart, and Angela Rita Armiento. 2020. "Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels" International Journal of Molecular Sciences 21, no. 23: 9195. https://doi.org/10.3390/ijms21239195
APA StyleHasler, J., Hatt, L. P., Stoddart, M. J., & Armiento, A. R. (2020). Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels. International Journal of Molecular Sciences, 21(23), 9195. https://doi.org/10.3390/ijms21239195