Table 2A.
Performance characteristics of total RNA isolated from the filtrates stored either at −80°C or Ambient (Room) Temperature.
| S.no | Time to process (days) | Volume (cc)a | A260/280 Ratiob | Nanodrop based total RNA (μg)c | Qubit based total RNA (μg)d | 18S rRNA copies/μg RNAe | TGFβ1 copies/μg RNAe | hsa-miR-26a copies/μg RNAe | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| −80°C | RT | −80°C | RT | −80°C | RT | −80°C | RT | −80°C | RT | −80°C | RT | |||
| 1 | 4 | 37.5 | 1.84 | 1.79 | 0.96 | 0.93 | 0.74 | 0.74 | 8.65E+09 | 6.50E+09 | 4.64E+04 | 1.99E+04 | 1.43E+06 | 1.69E+06 |
| 2 | 4 | 47.5 | 1.99 | 1.99 | 0.23 | 0.25 | 0.23 | 0.18 | 8.18E+08 | 1.13E+09 | 9.11E+03 | 5.38E+03 | 8.47E+05 | 9.36E+05 |
| 3 | 4 | 45 | 1.64 | 1.71 | 0.27 | 0.32 | 0.19 | 0.15 | 5.08E+08 | 8.31E+08 | 4.76E+03 | 3.44E+03 | 1.35E+06 | 8.51E+05 |
| 4 | 2 | 45 | 1.89 | 1.92 | 1.41 | 1.53 | 0.99 | 1.29 | 1.66E+10 | 2.01E+10 | 6.08E+04 | 4.88E+04 | 2.25E+06 | 3.04E+06 |
| 5 | 2 | 30 | 1.40 | 1.66 | 0.20 | 0.26 | 0.14 | 0.17 | 2.10E+08 | 7.70E+08 | 1.65E+02 | 3.62E+02 | 6.40E+05 | 4.31E+05 |
| 6 | 2 | 35 | 1.97 | 1.81 | 0.64 | 0.69 | 0.52 | 0.58 | 1.04E+09 | 2.33E+08 | 9.77E+02 | 1.67E+02 | 6.67E+05 | 6.02E+05 |
| 7 | 4 | 17.5 | 1.92 | 1.98 | 2.26 | 2.54 | 0.25 | 0.29 | 8.04E+08 | 1.75E+09 | 6.21E+03 | 2.12E+03 | 2.72E+06 | 2.07E+06 |
| 8f | 4 | 22.5 | 2.01 | 0.92 | 0.05 | 0.04 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
| 9 | 4 | 45 | 1.72 | 1.57 | 0.16 | 0.13 | 0.10 | 0.10 | 2.65E+08 | 1.94E+08 | 2.15E+03 | 4.64E+02 | 5.47E+05 | 8.34E+05 |
| 10 | 2 | 55 | 1.75 | 1.80 | 0.59 | 0.63 | 0.11 | 0.11 | 1.99E+09 | 2.46E+09 | 6.57E+03 | 8.11E+03 | 9.17E+05 | 7.85E+05 |
| 11 | 2 | 37.5 | 1.94 | 1.94 | 4.64 | 5.17 | 0.94 | 0.92 | 9.17E+08 | 1.14E+09 | 4.40E+03 | 4.02E+03 | 7.35E+05 | 6.08E+05 |
| 12 | 2 | 50 | 1.69 | 1.54 | 0.26 | 0.24 | 0.15 | 0.20 | 3.04E+08 | 5.86E+08 | 5.40E+02 | 2.52E+03 | 1.14E+06 | 7.96E+05 |
| Median | 41 | 1.87 | 1.80 | 0.43 | 0.48 | 0.23 | 0.20 | 8.18E+08 | 1.13E+09 | 4.76E+03 | 3.44E+03 | 9.17E+05 | 8.34E+05 | |
| 25th Percentile | 31 | 1.70 | 1.59 | 0.21 | 0.24 | 0.14 | 0.15 | 3.04E+08 | 5.86E+08 | 9.77E+02 | 4.64E+02 | 6.67E+05 | 6.08E+05 | |
| 75th Percentile | 47 | 1.96 | 1.94 | 1.30 | 1.38 | 0.74 | 0.74 | 1.99E+09 | 2.46E+09 | 9.11E+03 | 8.11E+03 | 1.43E+06 | 1.69E+06 | |
| Wilcoxon P value g | 0.57 | 0.04 | 0.30 | 0.24 | 0.08 | 0.58 | ||||||||
Urine samples from 12 kidney allograft recipients were processed using the ZR Urine RNA Isolation Kit™. One urine sample did not yield sufficient RNA for reverse transcription and was excluded from downstream analysis. The RNA containing filtrate were stored either at −80°C or at ambient temperature for 2 or 4 days prior to RNA isolation using Zymo-Spin™ IC Columns.
The volume of urine processed using the ZR Urine RNA Isolation Kit™.
A260/280 ratio was measured using the NanoDrop® ND-1000 spectrophotometer.
The amount of RNA measured using the NanoDrop® ND-1000 spectrophotometer.
The amount of total RNA measured using the Qubit fluorometer.
Absolute copy number of 18S rRNA, TGF-β1 mRNA, and hsa-miR-26a miRNA were measured with customized RT-qPCR assays using gene specific oligonucleotide pairs and TaqMan probes on 7500 Fast Real-Time PCR System (Applied Biosystems).
Total RNA yield from urine sample 8 was below detection limit for measurement using the Qubit Fluorometer and RNA was insufficient for reverse transcription to cDNA and measurement of 18S rRNA, TGF-β1 mRNA and miR-26a.
P values derived using Wilcoxon matched pairs signed-ranks test.