Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Groups
- Patients diagnosed with paroxysmal AF (n = 11);
- Patients with persistent AF (n = 14).
2.2. Clinical Examination of AF Patients
2.3. Blood Samples Collection and Treatment
2.4. The miRNA Purification from Blood Samples
2.5. Analysis of the Concentration and Purity of RNA
2.6. Selective Conversion of Mature miRNA into cDNA
2.7. The miRNA Expression Analysis
3. Results
3.1. Demographic and Clinical Overview of Study Donors
3.2. Concentration and Purity of miRNA
3.3. Optimization of miRNA Purification Procedure
3.4. miRNA Expression Analysis
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Wavelength (nm) | Substance |
---|---|
230 | EDTA, ethanol, polysaccharides |
260 | DNA, RNA |
280 | proteins |
320 | cellular debris |
Step | Time [min; s] | Temperature [°C] |
---|---|---|
Initial activation step | 15 min | 95 |
3-step cycling: | ||
Denaturation | 15 s | 95 |
Annealing | 30 s | 55 |
Extension | 34 s | 70 |
Cycle number | 45 | -xa |
Variable | Absolute Count and Percentage or Median and 25–75 Percentile or Mean ± SD | |
---|---|---|
Female sex | 11 (44%) | |
Age [years] | 56 (35–66) | |
Body mass index [kg/m2] | 28.44 ± 3.79 | |
CHA2DS2-VASc [pts] | 1 (1; 2) | |
EHRA score | 3 (2b; 3) | |
Mode of PVI | RF | 100% 0% |
Cryoablation | ||
AF type | Paroxysmal | 44% 56% |
Persistent | ||
Heart failure | 0 | |
Arterial hypertension | 16 (64%) | |
Diabetes | 4 (16%) | |
Coronary artery disease | 0 | |
Obesity | 11 (44%) | |
Hyperlipidemia | 15 (60%) | |
Smoking | 1 (4%) | |
Pharmacotherapy: | ||
Propafenone | 9 (36%) | |
Sotalol | 2 (8%) | |
Amiodarone | 9 (36%) | |
Anticoagulation: | ||
Dabigatran | 9 (36%) | |
Riwaroksaban | 15 (60%) | |
Apiksaban | 1 (4%) | |
Laboratory tests: | ||
Hemoglobin concentration [g/L] | 14.4 ± 2.4 | |
White blood cells [×109/L] | 6.8 ± 3.8 | |
Estimated glomerular filtration rate [mL/min/1.73 m2] | 86.8 ± 17.8 | |
Thyroid-stimulating hormone [uIU/mL] | 1.3 (0.9; 1.8) | |
Echocardiography: | ||
Left ventricular ejection fraction [%] | 55.4 ± 7.1 | |
Left atrial diameter [mm] | 39.2 ± 5.7 |
Study Design | Total RNA Concentration [ng/µL] | Absorbance Ratio A260/A280 | Absorbance Ratio A260/A230 |
---|---|---|---|
Control (n = 6) | 17.27 (7.30) | 1.42 | 0.27 |
Paroxysmal AF (n = 14) | 17.75 (7.11) | 1.31 | 0.28 |
Persistent AF group (n = 6) | 25.79 (19.50) | 0.64 | 0.43 |
Patient | Concentration of Total RNA [ng/µL] | Absorbance Ratio A260/A280 | Absorbance Ratio A260/A230 | Elution Step |
---|---|---|---|---|
C-6 | 15.7 | 0.4 | 0.24 | RNase-free water |
N-1 | 15.3 | 0.38 | 0.26 | |
N-2 | 25.8 | 0.65 | 0.42 | |
N-3 | 25.8 | 0.65 | 0.42 | |
N-4 | 12.4 | 0.3 | 0.2 | |
P-4 | 16.7 | 0.42 | 0.26 | |
C-6 | 8 | 0.2 | 0.12 | Tris-HCl |
N-1 | 9.2 | 0.23 | 0.15 | |
N-2 | 9.7 | 0.24 | 0.15 | |
N-3 | 26.5 | 0.66 | 0.5 | |
N-4 | 11.4 | 0.29 | 0.2 | |
P-4 | 6.4 | 0.16 | 0.11 |
Total RNA/miRNA Purification Package Kit | AF Donor | Total RNA Concentration (ng/µL) | Absorbance A260/280 Ratio | Absorbance A260/230 Ratio |
---|---|---|---|---|
miRNeasy Serum/Plasma Advanced kit | N-18 | 17.6 | 1.36 | 0.39 |
P-7 | 35.8 | 1.45 | 0.43 | |
miRNeasy Serum/Plasma kit | N-18 | 3.4 | 1.43 | 0.09 |
P-7 | 4.3 | 1.44 | 0.12 |
Amount of Plasma [µL] | Total RNA Concentration (ng/µL) | Absorbance Ratio A260/280 | Absorbance Ratio A260/230 |
---|---|---|---|
150 | 68.4 | 1.38 | 0.59 |
400 | 11.0 | 1.52 | 0.42 |
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Polak, M.; Wieczorek, J.; Botor, M.; Auguścik-Duma, A.; Hoffmann, A.; Wnuk-Wojnar, A.; Gawron, K.; Mizia-Stec, K. Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation. J. Clin. Med. 2024, 13, 1898. https://doi.org/10.3390/jcm13071898
Polak M, Wieczorek J, Botor M, Auguścik-Duma A, Hoffmann A, Wnuk-Wojnar A, Gawron K, Mizia-Stec K. Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation. Journal of Clinical Medicine. 2024; 13(7):1898. https://doi.org/10.3390/jcm13071898
Chicago/Turabian StylePolak, Mateusz, Joanna Wieczorek, Malwina Botor, Aleksandra Auguścik-Duma, Andrzej Hoffmann, Anna Wnuk-Wojnar, Katarzyna Gawron, and Katarzyna Mizia-Stec. 2024. "Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation" Journal of Clinical Medicine 13, no. 7: 1898. https://doi.org/10.3390/jcm13071898