Evaluation of Commercially Available Kits for Parallel DNA and microRNA Isolation Suitable for Epigenetic Analyses from Cell-Free Saliva and Salivary Extracellular Vesicles
Abstract
1. Introduction
2. Results
2.1. DNA Extraction from Cell-Free Saliva
Manufacturer | Abbreviation | Kit Name | Type of NA Isolated |
---|---|---|---|
Norgen-Biotek Corp., Thorold, ON, Canada | NG | Plasma/Serum RNA/DNA Purification Mini Kit | m/miRNA and DNA |
Qiagen., Hilden, Germany | QU | Qiagen AllPrep® DNA/RNA/miRNA Universal | m/miRNA and DNA |
Zymo Research Corporation., Tustin, CA, USA | ZY | Zymo ZR-Duet™ DNA/RNA MiniPrep Plus | m/miRNA and DNA |
Norgen-Biotek Corp., Thorold, ON, Canada | NG-1 | Norgen RNA/DNA Purification Kit | m/miRNA and DNA |
Norgen-Biotek Corp, Thorold, ON, Canada | NC-1 | Norgen Plasma/Serum Circulating and Exosomal RNA and DNA Purification Kit | m/miRNA and DNA |
Roche Diagnostics Deutschland GmbH., Mannheim, Germany | RH | Roche High Pure PCR Template Preparation kit | DNA only |
Norgen-Biotek Corp., Thorold, ON, Canada | NC | Norgen Plasma/Serum Cell Free Circulating DNA | DNA only |
2.2. miRNA Extraction from Cell-Free Saliva
Manufacturer | Abbreviation | Kit Name | Type of NA Isolated |
---|---|---|---|
Norgen-Biotek Corp., Thorold, ON, Canada | NG | Plasma/Serum RNA/DNA Purification Mini Kit | m/miRNA and DNA |
Qiagen, Hilden, Germany | QU | Qiagen AllPrep® DNA/RNA/miRNA Universal | m/miRNA and DNA |
Zymo Research Corporation., Tustin, CA, USA | ZY | Zymo ZR-Duet™ DNA/RNA MiniPrep Plus | m/miRNA and DNA |
Norgen-Biotek Corp., Thorold, ON, Canada | NG-1 | Norgen RNA/DNA Purification Kit | m/miRNA and DNA |
Norgen-Biotek Corp., Thorold, ON, Canada | NC-1 | Norgen Plasma/Serum Circulating and Exosomal RNA and DNA Purification Kit | m/miRNA and DNA |
Norgen-Biotek Corp., Thorold, ON, Canada | NT | Norgen Total RNA Purification Kit | m/miRNA only |
Exqion, Vedbæk, Denmark | EX | Exqion miRCURY™ RNA Isolation Kit—Cell & Plant | m/miRNA only |
Qiagen, Hilden, Germany | QA | miRNeasy Serum/Plasma Advanced Kit | m/miRNA only |
Norgen-Biotek Corp., Thorold, ON, Canada | NM | Plasma/Serum RNA Purification Mini Kit | m/miRNA only |
2.3. Evaluation of Commercial Kits for Extracellular Vesicle Isolation from Saliva
Manufacturer | Kit Code | Kit Name | Principle |
---|---|---|---|
Qiagen, Hilden, Ger-many | MR | miRCURY® Exosome Serum/Plasma Kit | Precipitation |
101 Bio., Mountain View, CA, USA | PE | PureExo® Exosome Isolation kit | Precipitation |
HansaBioMed Life Sciences., Tallinn, Estonia. | HI | Immunobeads for Overall exosomes capture from biological fluids | Immuno bead-based |
Norgen-Biotek Corp., Thorold, ON, Canada | NE | Norgen plasma serum exosomes Purification kit | Column Purification |
Tymora Analytical Operations, West Lafayette, IN, USA | EV | EVtrap magnetic capture of extracellular vesicles (EVs) | EV capture via magnetic beads |
3. Discussion
4. Materials and Methods
4.1. Saliva Donors and Collection
4.2. DNA and miRNA Extraction from Cell Free Saliva
4.3. Extracellular Vesicle Preparation and Isolation
4.4. DNA and RNA Concentration Measurements
4.5. DNA and miRNA Quantification via qPCR
4.6. Data Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Primer Name | Forward Primer Reverse Primer | Product Length | Cycles |
---|---|---|---|
H19 | TGG GCC GCA GTG CCT CGT GGG GGG CGA AGC GGC CAC GGG AG | 101 | 45 |
SNRPN | GTC CCC CAT CCG CCC CCA ACT G CCC ACT GCG GTT ACC CCG CAT GCT C | 105 | 45 |
JUB | GGC ATT GCT CTG CCC ATA GAT GCC TTT G GGA ATC CCT GGT TTT GAC CTG GGG GA | 101 | 45 |
TBP | GGC CCG CGG CTC TGT GCG GTG TCG GAT CCG CAG GCG CAG | 105 | 45 |
PCR-Reaction | 1 Reaction [µL] | ||
---|---|---|---|
AD | 5.716 | ||
10x Taq-buffer with MgCl (Qiagen) | 1.2 | ||
dNTP (2 mM) | 0.96 | ||
Eva Green (20x) (Biotium, Fremont, CA, USA) | 0.168 | ||
Primer fw (10 µM) | 0.24 | ||
Primer rev (10 µM) | 0.24 | ||
HotStar Taq Polymerase (Qiagen) | 0.072 | ||
Sample | 2 | ||
∑ qPCR-Program | 12 Temperature | Time | |
Activation | 5 min | ||
Denaturation | 95 °C | 40 s | |
Annealing | 65 °C | 40 s | 45x |
Elongation | 72 °C | 1 min 20 s | |
Final elongation | 72 °C | 7 min |
miRNA | Forward Primer Universal Reverse Primer |
---|---|
miR-16 | CGC GCT AGC AGC ACG TAA AT GTG CAG GGT CCG AGG T |
miR-21 | GCC CGC TAG CTT ATC AGA CTG ATG GTG CAG GGT CCG AGG T |
miR-30c-5p | AGC CGC CTG TAA ACA TCC TAC ACT GTG CAG GGT CCG AGG T |
miR-16 | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGCCAA |
miR-21 | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA |
miR-30c-5p | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGAG |
Master Mix | 1 Reaction [µL] | ||
---|---|---|---|
AD | 5.66 | ||
10x buffer | 1.5 | ||
100 mM dNTP | 0.15 | ||
RNAse Inhibitor (20 U/µL) | 0.19 | ||
Mutiscripte RT | 1 | ||
RT-loop Primer (20 nM) | 1.5 | ||
Master mix | 10 | ||
Sample (RNA) | 5 | ||
total ∑ | 15 | ||
RT-Program | Temperature | Time | |
Activation | 16 °C | 30 min | |
Denaturation | 20 °C | 30 s | |
Annealing | 42 °C | 30 s | 60x |
Elongation | 50 °C | 1 s | |
Final elongation | 85 °C | 5 min | |
Hold | 4 °C | hold |
PCR-Reaction | 1 Reaction [µL] | ||
---|---|---|---|
AD | 5.716 | ||
10 x Taq-buffer with MgCl (Qiagen) | 1.2 | ||
dNTP (2 mM) | 0.96 | ||
Eva Green (20x) (Biotium) | 0.168 | ||
Primer uni Rev (100 µM) | 0.084 | ||
HotStar Taq Polymerase (Qiagen) | 0.072 | ||
Primer fw(10 µM) | 1.8 | ||
Sample | 2 | ||
∑ qPCR-Program | 12 Temperature | Time | |
Activation | 05 min | ||
Denaturation | 95 °C | 40 s | |
Annealing | 60 °C | 40 s | 45x |
Elongation | 72 °C | 1 min 20 s | |
Final elongation | 72 °C | 7 min |
Appendix B
Nanodrop | Cell Free Saliva DNA Gene Markers | ||||
---|---|---|---|---|---|
Picogreen | SNRPN | JUB | TBP | H19 | |
QU x NG | <0.001 | <0.001 | <0.001 | 0.999 | 0.057 |
ZY x NG | <0.001 | <0.001 | <0.001 | 0.999 | 0.008 |
NG-1 x NG | <0.001 | <0.001 | 0.675 | 0.998 | 0.178 |
NC-1 x NG | <0.001 | 0.141 | 0.841 | 0.999 | 1.000 |
RH x NG | <0.001 | 0.008 | <0.001 | 0.999 | <0.001 |
NC x NG | <0.001 | 0.024 | 0.002 | 0.940 | 0.008 |
ZY x QU | 0.356 | 0.990 | 0.003 | 0.990 | 0.924 |
NG-1 x QU | 0.111 | 0.005 | <0.001 | 0.987 | 0.993 |
NC-1 x QU | <0.001 | <0.001 | <0.001 | 0.999 | 0.108 |
RH x QU | 0.481 | <0.001 | <0.001 | 0.999 | <0.001 |
NC x QU | 0.166 | <0.001 | <0.001 | 0.866 | 0.924 |
NG-1 x ZY | 0.002 | 0.005 | <0.001 | 0.999 | 0.599 |
NC-1 x ZY | <0.001 | <0.001 | <0.001 | 0.999 | 0.016 |
RH x ZY | 1.000 | <0.001 | 0.949 | 0.999 | <0.001 |
NC x ZY | 0.0034 | <0.001 | <0.001 | 0.998 | 0.999 |
NC-1 x NG-1 | <0.001 | <0.001 | 0.118 | 0.999 | 0.305 |
RH x NG-1 | 0.003 | <0.001 | <0.001 | 0.999 | <0.001 |
NC x NG-1 | 0.999 | 0.203 | 0.035 | 0.998 | 0.599 |
RH x NC-1 | <0.001 | 0.666 | <0.001 | 0.999 | <0.001 |
RH x NG-1 | <0.001 | <0.001 | <0.001 | 0.977 | 0.016 |
NC x NG-1 | 0.005 | <0.001 | <0.001 | 0.965 | <0.001 |
RH x NC-1 | <0.001 | <0.001 | <0.001 | 0.999 | 0.057 |
NC x NC-1 | <0.001 | <0.001 | <0.001 | 0.999 | 0.008 |
NC x RH | <0.001 | <0.001 | 0.675 | 0.998 | 0.178 |
Appendix C
Nanodrop | Cell Free Saliva miRNA Gene Markers | |||
---|---|---|---|---|
Riobgreen | miR16 | miR21-5p | miR30c-5p | |
QU x NG | <0.01 | <0.001 | <0.001 | <0.001 |
ZY x NG | <0.01 | 0.999 | <0.001 | <0.001 |
NG-1 x NG | <0.01 | 0.559 | 0.979 | 0.928 |
NC-1 x NG | <0.01 | <0.001 | 0.565 | 0.875 |
NT x NG | 0.998 | 0.999 | 0.888 | 0.106 |
EX x NG | 0.806 | <0.001 | 0.001 | 0.005 |
QA x NG | 0.788 | <0.001 | <0.001 | <0.001 |
NM x NG | 0.720 | 0.309 | 0.169 | <0.001 |
ZY x QU | 0.969 | <0.001 | 0.269 | 0.479 |
NG-1 x QU | 0.999 | 0.00162 | <0.001 | <0.001 |
NC-1 x QU | 0.979 | 0.999 | <0.001 | <0.001 |
NT x QU | <0.01 | <0.001 | <0.001 | 0.01249 |
EX x QU | 0.0104 | <0.001 | <0.001 | <0.001 |
QA x QU | <0.01 | <0.001 | <0.001 | <0.001 |
NM x QU | 0.0145 | <0.001 | <0.001 | 0.724 |
NG-1 x ZY | 0.999 | 0.690 | <0.001 | <0.001 |
NC-1 x ZY | 0.504 | <0.001 | <0.001 | <0.001 |
NT x ZY | <0.01 | 0.999 | 0.00137 | <0.001 |
EX x ZY | 0.089 | <0.001 | <0.001 | <0.001 |
QA x ZY | <0.01 | <0.001 | <0.001 | <0.001 |
NM x ZY | 0.119 | 0.220 | 0.025 | 0.024 |
NC-1 x NG-1 | 0.843 | 0.002 | 0.126 | 0.999 |
NT x NG-1 | <0.01 | 0.676 | 0.999 | 0.657 |
EX x NG-1 | 0.0279 | <0.001 | <0.001 | <0.001 |
QA x NG-1 | <0.01 | <0.001 | <0.001 | <0.001 |
NM x NG-1 | 0.0381 | 0.007 | 0.665 | 0.010 |
NT x NC-1 | <0.01 | <0.001 | 0.063 | 0.7461 |
EX x NC-1 | <0.01 | <0.001 | 0.060 | <0.001 |
QA x NC-1 | <0.01 | <0.001 | <0.001 | <0.001 |
NM x NC-1 | <0.01 | <0.001 | 0.00345 | 0.014 |
EX x NT | 0.404 | <0.001 | <0.001 | <0.001 |
QA x NT | 0.989 | <0.001 | <0.001 | <0.001 |
NM x NT | 0.324 | 0.229 | 0.861 | 0.310 |
QA x EX | 0.092 | <0.001 | <0.001 | <0.001 |
NM x EX | 1 | <0.001 | <0.001 | <0.001 |
NM x QA | 0.068 | <0.001 | <0.001 | <0.001 |
Appendix D
Nanodrop | DNA Gene Marker | Nanodrop | miRNA Gene Marker | ||||||
---|---|---|---|---|---|---|---|---|---|
Picogreen | SNRPN | JUB | TBP | H19 | Ribogreen | miR16 | miR21-5p | miR30c-5p | |
HI x EV | <0.001 | 0.820 | 0.604 | 0.990 | 0.747 | 0.013 | 0.820 | 0.604 | 0.990 |
MR x EV | <0.001 | <0.001 | <0.001 | 0.001 | <0.001 | <0.001 | <0.001 | <0.001 | 0.001 |
NE x EV | <0.001 | 0.998 | 0.860 | 0.528 | 1.000 | <0.001 | 0.998 | 0.860 | 0.528 |
PE x EV | <0.001 | 0.002 | 0.010 | 0.128 | <0.001 | 0.002 | 0.002 | 0.010 | 0.128 |
MR x HI | <0.001 | 0.000 | 0.007 | <0.001 | <0.001 | <0.001 | 0.000 | 0.007 | <0.001 |
NE x HI | 1.000 | 0.669 | 0.987 | 0.779 | 0.793 | <0.001 | 0.669 | 0.987 | 0.779 |
PE x HI | 0.367 | 0.011 | 0.093 | 0.064 | 0.005 | 0.815 | 0.011 | 0.093 | 0.064 |
NE x MR | <0.001 | <0.001 | 0.003 | <0.001 | <0.001 | 0.000 | <0.001 | 0.003 | <0.001 |
PE x MR | <0.001 | 0.065 | 0.463 | 0.087 | 0.060 | <1 × 10−4 | 0.065 | 0.463 | 0.087 |
PE x NE | 0.306 | 0.001 | 0.044 | 0.011 | 0.002 | <1 × 10−4 | 0.001 | 0.044 | 0.011 |
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Yousaf, I.; Kegler, U.; Hofner, M.; Noehammer, C. Evaluation of Commercially Available Kits for Parallel DNA and microRNA Isolation Suitable for Epigenetic Analyses from Cell-Free Saliva and Salivary Extracellular Vesicles. Int. J. Mol. Sci. 2025, 26, 6365. https://doi.org/10.3390/ijms26136365
Yousaf I, Kegler U, Hofner M, Noehammer C. Evaluation of Commercially Available Kits for Parallel DNA and microRNA Isolation Suitable for Epigenetic Analyses from Cell-Free Saliva and Salivary Extracellular Vesicles. International Journal of Molecular Sciences. 2025; 26(13):6365. https://doi.org/10.3390/ijms26136365
Chicago/Turabian StyleYousaf, Iqra, Ulrike Kegler, Manuela Hofner, and Christa Noehammer. 2025. "Evaluation of Commercially Available Kits for Parallel DNA and microRNA Isolation Suitable for Epigenetic Analyses from Cell-Free Saliva and Salivary Extracellular Vesicles" International Journal of Molecular Sciences 26, no. 13: 6365. https://doi.org/10.3390/ijms26136365
APA StyleYousaf, I., Kegler, U., Hofner, M., & Noehammer, C. (2025). Evaluation of Commercially Available Kits for Parallel DNA and microRNA Isolation Suitable for Epigenetic Analyses from Cell-Free Saliva and Salivary Extracellular Vesicles. International Journal of Molecular Sciences, 26(13), 6365. https://doi.org/10.3390/ijms26136365