Magnetic-Bead and Spin-Column-based RNA purification systems extracted equivalent whole-blood RNA quantity, quality and purity.
The quantity, quality and purity of RNA extracted were evaluated using two standard RNA isolation systems (18): spin-column-based (PAXgene® QIAGEN/BD; Tempus™ Applied Biosystems) or magnetic-bead-based (MagMAX™ Life Technologies; compatible with either PAXgene® or Tempus™ collection tube) systems. RNA concentration (ng/µL) and purity (A260/A280) were evaluated by spectroscopic quantification using NanoPhotometer® N60 (Implen, München, Germany). RNA integrity number (RIN) was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA yield (ng) was normalized to whole blood volume collected in each blood RNA collection system (i.e., 2.5 mL in PAXgene®, vs. 3 mL in Tempus™). All RNA samples were extracted fresh post-collection for the comparison of RNA isolation protocols.
We found the RNA isolation protocol did not affect the amount of total RNA extracted, the RNA quality, or the purity of RNA (Normalized total RNA: p = 0.875, RIN: p = 0.124, A260/A280: p = 0.101, MagMAX™ vs. columns, unpaired t-test) (Fig. S1). Additionally, all samples had RIN of > 7.0 and an A260/A280 ratio between 1.98–2.15, suggesting recovery of high-quality and high-purity RNA from both RNA isolation systems. We found RNA yields obtained from the Tempus™ tubes were significantly lower than those obtained with PAXgene® tubes (p = 0.008, unpaired t-test), suggesting that the PAXgene® tubes give higher RNA yields than Tempus™ when extracted in optimal laboratory conditions (Fig. S1). Given the high quality of the extracted RNA and the advantages of extracting in a 96-well-plate format (6), magnetic bead-based (MagMAX™) RNA purification system compatible with PAXgene® and Tempus™ systems were used for the rest of the extractions.
A higher quantity of RNA was obtained using Tempus™ Blood RNA Tubes in suboptimal tropical conditions.
Next, we compared the quantity, quality, and purity of RNA extracted from whole-blood samples stored in suboptimal tropical conditions collected in PAXgene® and Tempus™ tubes. To simulate suboptimal tropical conditions, matched whole-blood samples were collected in either PAXgene® or Tempus™ tubes and stored at different temperatures (25, 30, 35 or 40°C) for different lengths of time (0, 1, 5, 7 or 10 days) before storage at -80°C for later extraction. These samples were compared to matched samples immediately frozen at -80°C for later extraction (D1/Control) or unmatched samples collected and processed post-collection immediately in optimal laboratory conditions (D0/Fresh). The tube type had no effect on RNA quantity (i.e., normalized RNA concentration) when extracted in Fresh (p = 0.065, paired t-test) or Control (p = 0.274, paired t-test) conditions (Fig. 2A). However, we found that tube type significantly affected normalized RNA concentration (p < 0.0001; Table 1, Model 1) in samples subjected to suboptimal tropical conditions.
To investigate these data further, we applied three multiple linear regression models to evaluate the effects of explanatory variables (i.e., tube type, storage times, temperature, and biological subject) on normalized RNA concentrations, A260/A280 ratios and RIN values (Table 1). Model 1 explained 76% of the variation in extracted RNA quantity (R2 = 0.764, p < 2.2×10− 16). Tempus™ tubes had a significant effect on the RNA yield (p = 1.15e-06), and temperature variation in tube type significantly impacted RNA yield (p = 0.0003; Table 1, Model 1). Additionally, we applied separate linear models to each temperature condition to explore the effect of the temperature on the tube type (Table S3). We found that the concentration of RNA extracted from whole-blood collected in Tempus™ tubes was significantly greater than for PAXgene® tubes at all evaluated temperatures [p = 7.16e-06 (25°C); p = 0.0004 (30°C); p = 0.033 (35°C); p = 0.023 (40°C), Table S3]. As a secondary measurement of RNA concentration, we obtained the RNA concentration readings from Agilent Bioanalyzer. Similarly, Tempus™ tubes gave higher RNA yields when measured with Agilent Bioanalyzer (p = 0.001; Table S4). Interestingly, RNA concentration measurements by spectrophotometer and bioanalyzer were more strongly correlated in Tempus™ than in PAXgene® tubes [p = 0.0002, R2 = 0.270 (PAXgene®); p = 9.3×10− 9, R2 = 0.518 (Tempus™)]; Fig. 2B), suggesting that tube-specific contents influence concentration measurements.
Table 1
Multiple linear regression models. Model 1 on normalized RNA concentration (ng/µL), Model 2 on A260/A280 ratios and Model 3 on RIN (expressed as log2 RIN)
Model 1 = RNA concentration (ng/µL)
|
Explanatory variable
|
Estimate
|
Std. Error
|
t value
|
p value
|
(Intercept)
|
66.230
|
78.853
|
0.84
|
0.404
|
Tube type: Tempus
|
254.894
|
47.339
|
5.384
|
1.15e-06***
|
Days
|
5.049
|
9.997
|
0.505
|
0.615
|
Temperature
|
-0.106
|
2.358
|
-0.045
|
0.964
|
Tube type Tempus: Days
|
-5.859
|
3.342
|
-1.753
|
0.084
|
Tube type Tempus: Temperature
|
-4.650
|
1.228
|
-3.786
|
0.0003*
|
Days: Temperature
|
-0.170
|
0.299
|
-0.57
|
0.571
|
Adjusted R-squared: 0.764 (p value: <2.2e-16)
|
Model 2 = A260/A280
|
Explanatory variable
|
Estimate
|
Std. Error
|
t value
|
p value
|
(Intercept)
|
2.181
|
0.102
|
21.466
|
< 2e-16***
|
Tube type: Tempus
|
0.161
|
0.061
|
2.638
|
0.011*
|
Days
|
0.008
|
0.013
|
0.606
|
0.547
|
Temperature
|
0.000
|
0.003
|
-0.085
|
0.933
|
Tube type Tempus: Days
|
-0.008
|
0.004
|
-1.951
|
0.055
|
Tube type Tempus: Temperature
|
-0.005
|
0.002
|
-3.075
|
0.003**
|
Days: Temperature
|
0.000
|
0.000
|
-0.567
|
0.572
|
Adjusted R-squared:0.565 (p value: 1.458e-10)
|
Model 3 = log2(RIN)
|
Explanatory variable
|
Estimate
|
Std. Error
|
t value
|
p value
|
(Intercept)
|
4.905
|
0.572
|
8.581
|
3.45e-12***
|
Tube type: Tempus
|
0.338
|
0.343
|
0.985
|
0.328
|
Days
|
-0.148
|
0.072
|
-2.047
|
0.045*
|
Temperature
|
-0.082
|
0.017
|
-4.777
|
1.10e-05***
|
Tube type Tempus: Days
|
-0.020
|
0.024
|
-0.836
|
0.407
|
Tube type Tempus: Temperature
|
0.002
|
0.009
|
0.236
|
0.814
|
Days: Temperature
|
0.002
|
0.002
|
1.057
|
0.295
|
Adjusted R-squared: 0.797 (p value: <2.2e-16)
|
Independent multiple linear regression models: Model 1 on normalized RNA concentration, Model 2 on A260/A280 ratios and Model 2 on log2(RIN) values as dependent variable and tube types, days, temperatures and subjects as the independent variables. *** p < 0.001, **p < 0.01, *p < 0.05 |
When considering RNA purity, all extracted RNA samples had A260/A280 ratios >2, regardless of tube type, indicating a high purity under all test conditions (Fig. 2A, middle panel). There was no difference in A260/A280 ratios between tube type for RNA extracted from Fresh and Control RNA samples ([p=0.480, (Fresh); p=0.111, (Control), paired t-test]. However, at tropical storage and temperature conditions, the tube type (p=0.011) and the storage temperature on tube type (p=0.003) had significant effects on A260/A280 ratios as per the multiple linear regression model (Table 1, Model 2). Linear models further indicated that the higher temperatures decreased purity as evidenced by A260/A280 ratio [30°C (p=0.007), 35°C (p=0.011) and 40°C (p=4.89e-05), Table S3]. Taken together, these data demonstrated that higher RNA yields are extracted from Tempus™ blood tubes compared to PAXgene® tubes in suboptimal tropical conditions. These data also showed that RNA yields significantly decreased with increasing temperature in both PAXgene® and Tempus™ tubes.
A higher quality of RNA was obtained using PAXgene® tubes in optimal laboratory conditions.
We determined if high RNA quality was preserved using PAXgene® or Tempus™ tubes in suboptimal tropical conditions and compared the RNA quality with samples extracted in optimal laboratory conditions. RIN values declined over time and temperature, irrespective of the whole-blood collection tube (Fig. 2A). PAXgene® had significantly higher RIN values in ‘Fresh’ (p=0.013, paired t-test) and ‘Control’ conditions (p=0.001, paired t-test) compared to Tempus™. The electropherograms showed comparable results for different conditions applied on PAXgene® and Tempus™ systems (Fig. 2C). Ribosomal RNA bands were clearly visible in ‘Fresh’ extractions and ‘Control’ samples. Most RNA eluates stored at room temperature (25°C) for 5-7 days obtained RIN values around 5–6 with visible 18s and 28s bands. In contrast, RNA stored at 40°C did not show distinct rRNA banding. These data demonstrated that higher quality RNA was obtained with PAXgene® tubes (compared to Tempus™) when RNA was extracted post-collection immediately or when samples were maintained at the optimal storage conditions as recommended by the manufacturers.
The highest quality RNA was obtained using Tempus™ Blood RNA tubes in suboptimal tropical conditions.
A multiple linear regression model was built to explore the effects of suboptimal tropical conditions on RNA integrity to determine the effect of tube type, storage temperature, and storage time on RNA integrity (measured by RIN values) (Table 1, Model 3). Model 3 explained approximately 79.7% (R2 = 0.797) of the variation in RIN values. RIN values decreased significantly over time (p = 0.045), decreasing by 0.862 per day (Model 3, Days Estimate =-0.148; e− 0.148=0.862). RIN values also decreased significantly with the increasing of temperature (p < 0.0001), decreasing by 0.921 (Model 3, Temperature Estimate= -0.082; e− 0.082=0.921) for each degree Celsius (°C) increase in temperature. These data demonstrated that RNA integrity significantly decreased with the length of storage time and temperature.
The impact of storage time on RNA integrity (RIN values) was also analyzed at each temperature for each tube type (Table S3). RIN values were significantly higher in Tempus™ than PAXgene® tubes at 30°C (p = 0.0001) and 35°C (p = 0.001). Although, no significant differences in RIN at 25°C (p = 0.787) and 40°C (p = 0.399) for tube types (Fig. 2A). In agreement with previously published studies (9, 22), these results demonstrated that RNA integrity is temperature-sensitive, and both tube types produced low-quality RNA at increased storage times and temperatures. Nevertheless, our data suggest that Tempus™ tubes may provide better RNA integrity (higher RIN values) under certain suboptimal tropical conditions compared to PAXgene® tubes.
Tempus™ tubes maintain mRNA integrity across suboptimal tropical conditions.
In order to validate our RNA quality measurements, we quantified mRNA and rRNA extracted from PAXgene® and Tempus™ tubes using RT-qPCR. We tested the relative mRNA abundance of two human reference genes, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), and one rRNA transcript 18S ribosomal RNA (18s). The RNA concentration of all samples was normalized at pre-cDNA synthesis (i.e., at 30 ng/µL). Hence, an increasing cycle threshold (Ct) value indicated a decreasing relative transcript quality rather than abundance (23). We tested RT-qPCR primer sets designed to amplify different-sized fragments of the same target gene (i.e., amplicons between 100–300 bp) and differences in the relative RNA quality (e.g., increased Ct values) would be expected to be intensified when assaying genes with primers amplifying larger amplicons.
There was no significant difference in the mean Ct values between the tube types for Control samples from smaller amplicons (p = ns, paired t-test: D1/Control, 100–200 bp) or with larger amplicons (p = ns, paired t-test: D1/Control, 200–300 bp, Fig. 3). In addition, we found no statistically significant difference between matched ‘Fresh’ and ‘Control’ samples between PAXgene® and Tempus™ tubes (Fig. S4). However, the Ct value varied significantly at tropical storage conditions across all three tested genes across short (100–200 bp) and medium amplicons (200–300 bp) (Fig. 3). Tempus™ tubes maintained significantly higher transcript stability, as indicated by lower Ct values obtained for three tested genes compared to PAXgene® tubes at suboptimal tropical conditions (Table S5).
Multiple comparison testing found that the 18s rRNA Ct values were not statistically significantly influenced by incubation temperature or duration when rRNA was collected in either PAXgene® or Tempus™ tubes. In contrast, mRNA (SDHA and TBP) collected in PAXgene® tubes were significantly impacted by storage time and temperature compared to Tempus™ tubes (Table S6).
We showed that the RNA degraded samples, as indicated by the decreasing level of RIN had higher Ct values (Fig. 4A). The Ct shifted towards higher cycle numbers for SDHA with larger amplicons than short and medium length amplicons, which was much more evident in PAXgene® tubes than in Tempus™ (Fig. 4A). These results indicated that relative overall stability in terms of mRNA expression levels were maintained in Tempus™ compared to PAXgene® tubes. A similar relationship between RIN and Ct values was observed for TBP (Fig. S5). However, Figure S5 clearly shows that both tube types had Ct < 30 for all product lengths for 18s, suggesting both PAXgene® and Tempus™ tubes preserved rRNA at suboptimal tropical conditions. As indicative of decreasing relative transcript quality, increasing Ct values were validated by correlating change in Ct values with RIN. We considered the change in Ct values (∆Ct) as the difference between samples collected under suboptimal tropical conditions and the mean of the ‘Control’ samples. Strong statistically significant correlations were found between ∆Ct and RIN for all tested genes (Fig. 4B). These negative correlations indicated that with the decreasing RIN values, the ∆Ct of 200-300bp amplicons increased, thus validating the use of RT-qPCR to assess the quality of the RNA. Taken together, these data demonstrate that Tempus™ collection tubes better maintain mRNA stability in suboptimal tropical conditions even if RIN is significantly decreasing.
To test if the presence of PCR inhibitors, which are often co-extracted from whole blood (e.g., haemoglobin, lactoferrin, anticoagulants, etc.) (24, 25), could have contributed to these results, RT-qPCR was performed on a log2 serial dilution of undiluted extraction eluent. We considered that a trendline gradient of Ct values relative to the dilution greater than − 3.3 (i.e., E’ < 100%) is indicative of the presence of PCR inhibitors (24). There was no apparent effect of inhibitors in both PAXgene® or Tempus™ tubes when the samples were diluted below 60ng/uL (Fig. S3). These data demonstrated that our findings were unlikely to be a consequence of inhibitors present in the RT-qPCR reaction.
In summary, our data showed that Tempus™ tubes maintained a higher RNA quantity and integrity comparatively to PAXgene® tubes when RNA is stored in suboptimal tropical conditions. Furthermore, Tempus™ tubes maintained stability of mRNA in conditions where RNA samples were heavily degraded as indicated by RIN. Taken together, this study establishes that the Tempus™ blood RNA collection system resulted in a better quality of RNA and enhanced stability of mRNA when whole blood samples are stored under suboptimal tropical conditions.