Human blood RNA stabilization in samples collected and transported for a large biobank
© Duale et al.; licensee BioMed Central Ltd. 2012
Received: 27 April 2012
Accepted: 13 September 2012
Published: 18 September 2012
The Norwegian Mother and Child Cohort Study (MoBa) is a nation-wide population-based pregnancy cohort initiated in 1999, comprising more than 108.000 pregnancies recruited between 1999 and 2008. In this study we evaluated the feasibility of integrating RNA analyses into existing MoBa protocols. We compared two different blood RNA collection tube systems – the PAXgene™ Blood RNA system and the Tempus™ Blood RNA system - and assessed the effects of suboptimal blood volumes in collection tubes and of transportation of blood samples by standard mail. Endpoints to characterize the samples were RNA quality and yield, and the RNA transcript stability of selected genes.
High-quality RNA could be extracted from blood samples stabilized with both PAXgene and Tempus tubes. The RNA yields obtained from the blood samples collected in Tempus tubes were consistently higher than from PAXgene tubes. Higher RNA yields were obtained from cord blood (3 – 4 times) compared to adult blood with both types of tubes. Transportation of samples by standard mail had moderate effects on RNA quality and RNA transcript stability; the overall RNA quality of the transported samples was high. Some unexplained changes in gene expression were noted, which seemed to correlate with suboptimal blood volumes collected in the tubes. Temperature variations during transportation may also be of some importance.
Our results strongly suggest that special collection tubes are necessary for RNA stabilization and they should be used for establishing new biobanks. We also show that the 50,000 samples collected in the MoBa biobank provide RNA of high quality and in sufficient amounts to allow gene expression analyses for studying the association of disease with altered patterns of gene expression.
KeywordsTempus tubes PAXgene tubes Cord blood Quality control RNA stabilization Biobank Blood RNA Gene expression
The Norwegian Mother and Child Cohort Study (MoBa) is a nation-wide population-based pregnancy cohort initiated in 1999 [1, 2]. The MoBa holds more than 108,000 pregnancies recruited between 1999 and 2008; the last child to be included was born in June 2009. In addition to detailed questionnaire data, biological materials from the mother, the father and the child (umbilical cord blood) in the form of whole blood and plasma, have been collected and are stored in a biobank at the Norwegian Institute for Public Health (NIPH) together with extracted DNA, for future use . The DNA is routinely extracted from the blood samples from each participant and frozen in aliquots. In 2005 funds were obtained from the US National Institutes of Health through the Autism Birth Cohort to initiate RNA collection for the purpose of gene expression analyses. The challenge in implementing RNA collection was to devise a new protocol that would be feasible in maternity units and during transportation - and at the same time the existing routines and logistics of the cohort should be adhered to as closely as possible. There are several reports on the ex vivo instability of RNA transcripts [3–5], and it is therefore very important to stabilize blood RNA during sample collection, transport, and storage , in order to obtain reproducible gene expression results.
The overall goal of this study was to establish a practical protocol for sampling, handling, transportation and storage of adult and cord blood RNA. On this background, we systematically compared the RNA quality, quantity, and the RNA transcript stability of two commercially available RNA stabilizing technologies PAXgene™ Blood RNA system (PreAnalytiX, QIAGEN/BD, Hombrechtikon, Switzerland), and Tempus™ Blood RNA system (Applied Biosystems, Foster City, CA). We then investigated the effects of transportation and blood volumes on the RNA transcript stability of six selected genes (CDKN1A, FOS, IL8, MYC, IL1B, and TP53) using quantitative real-time PCR (RT-qPCR). These genes were selected based on literature search [7, 8].
Results and discussion
In MoBa, the combination of biological specimens and questionnaire data on lifestyle and exposures provide unique possibilities to study the effects of many factors of relevance for pregnancy outcomes and health . Blood-based large biobanks such as MoBa, and multi-center studies, increasingly incorporate gene expression profiling studies in order to get insight into the biological mechanisms triggered by gene-environment interactions and disease. However, collection, transportation, processing and storage of blood samples may represent logistical and technical challenges [9, 10]. Therefore, establishment of robust and standardized protocols is a prerequisite in order to reduce the impact which preanalytical sample handling may exert on RNA quality and stability. This is particularly important when collecting blood samples for large-scale biobanks, where the associated costs are very high.
In this study, we investigated important input factors for large-scale collection of blood from adults and children (umbilical cord blood). Although high-quality RNA can be prepared using fresh blood that is processed immediately, this option is hardly realistic when sampling for a large-scale biobank.
Comparison of two RNA stabilizing systems, PAXgene and Tempus
Comparison of blood samples collected in PAXgene and in Tempus tubes
A) Adult blood
Storage at RT (days)
OD 260/280 ratio
OD 260/230 ratio
Number of tubes
8.0 ± 0.3
2.12 ± 0.02
1.56 ± 0.31
8.0 ± 0.3
2.10 ± 0.03
1.56 ± 0.62
8.2 ± 0.3
2.12 ± 0.02
1.37 ± 0.73
8.2 ± 0.3
2.09 ± 0.03
1.42 ± 0.70
8.10 ± 0.3
2.11 ± 0.02
1.48 ± 0.61*
8.0 ± 0.3
2.12 ± 0.07
2.01 ± 0.14
8.2 ± 0.3
2.12 ± 0.07
2.01 ± 0.14
8.0 ± 0.3
2.14 ± 0.08
2.09 ± 0.08
8.2 ± 0.3
2.11 ± 0.04
2.08 ± 0.06
8.10 ± 0.3
2.12 ± 0.07
2.05 ± 0.11
B) Cord blood
PAXgene (n = 8)
Tempus (n = 7)
8.1 ± 0.2
8.0 ± 0.2
OD 260/280 ratio
2.1 ± 0.1
2.0 ± 0.3
OD 260/230 ratio
1.6 ± 0.5*
2.0 ± 0.8
The integrity and purity of RNA can be used to evaluate the quality of sample handling and processing. A RIN-value higher than five is considered as indicating good quality RNA whereas RIN-values above eight are considered ideal for downstream applications [4, 5]. When we investigated the RNA quality, we observed similar and comparable average OD 260/280 ratios and average RIN values for all adult and cord blood samples collected in Tempus and PAXgene tubes (p > 0.05), but the average OD 260/230 ratio for the samples collected in the PAXgene tubes was lower (p < 0.05) than for samples collected in Tempus tubes (Table 1). It has previously been reported that OD 260/230 ratios are higher for Tempus tubes compared with PAXgene tubes . The observed differences in the average OD 260/230 ratio between the two systems may be due to the PAXgene elution buffer which may contain high salt contents. Storage of PAXgene and Tempus samples at room temperature for up to seven days had little effect on RNA quality, although a slight decrease in RNA yield (p < 0.05) was observed for adult samples collected in the PAXgene tubes at seven days of storage (Figure 2A). The RNAs isolated from both PAXgene and Tempus had average RIN-values of approximately eight, and average OD 260/A280 ratios were above 1.8 (Table 1). Based on these data, high-quality RNA could be extracted from blood samples stabilized with both PAXgene and the Tempus system.
The RNA transcript stability and potential alteration of the transcript level for four genes (CDKN1A, IL8, MYC, and TP53) were investigated by RT-qPCR (Figure 2B-D). Storage of both PAXgene and Tempus tubes at room temperature for up to seven days had some effect on RNA transcript stability; there were clear storage related changes in the transcript levels. For adult blood samples collected both in PAXgene and Tempus tubes, storage for up to seven days at room temperature had effects on the relative transcript levels for the four genes when compared to the day 0 samples (Figure 2B and C). Storage effects were more pronounced in samples collected in the PAXgene compared to the Tempus tubes. However, the observed transcript level changes were within ±2–fold for both types of tubes (Figure 2B and C). The non-normalized raw Cq-values for the four genes also illustrate similar trends (Additional file 1A and B). The differences in the Cq-values between stored and day 0 samples for each gene were very small. For cord blood samples, the average transcript levels for the four genes were comparable between the Tempus and the PAXgene tube samples, and the difference between the two systems (Figure 2D and Additional file 1C) was not significant (p > 0.05). Storage for up to 7 days exceeds the maximum period (3–5 days) as recommended by both manufacturers. The recommended storage time may be too strict, and a more flexible protocol – also giving RNA of acceptable quality - can more conveniently be implemented in multi-center studies.
Given the results of this initial investigation where samples collected in the Tempus tubes gave higher RNA yields with comparable quality when compared to PAXgene tubes, we selected the Tempus Blood RNA system for MoBa and began collecting cord blood in Tempus tubes in June 2005. Since then, approximately 50,000 samples have been collected and stored at −80°C.
Transportation QC: The effect of transportation on RNA quality and transcript stability
Figure 3B shows the relative transcript levels for the six genes. Transportation of the samples apparently affected the relative transcription levels of the genes. Samples sent to Kirkenes or Kristiansand showed the highest changes compared to the samples kept at NIPH (control samples) (Figure 3B). However, the observed transcript level changes were again within ± 2–fold. The non-normalized raw Cq-values for the six genes and the average raw Cq-values ± SD for each gene are presented in Additional file 2D. Differences in the Cq-values between transported samples and samples kept at NIPH for each gene were small. A significant finding is that higher RNA yields were obtained from transported samples compared to samples kept at NIPH (Figure 3A and Additional file 2C); the reason for this is unclear. Samples can be shaken during transportation due to mechanical movements and vibrations, and temperature fluctuations occur (Additional file 2C), which may affect the RNA extraction efficiency.
Overall, transportation of the samples by standard mail had some effects on RNA yield, quality and RNA transcript stability. Temperature fluctuations, mechanical movements or vibrations during the transportation of samples may have contributed to the differences (Figure 3 and Additional file 2). However, the observed differences between transported samples and samples kept at NIPH were minor, and the obtained RNA quality from transported samples was considered acceptable for many RNA assays. Our results therefore suggest that blood samples collected in Tempus tubes can be sent by standard mail, under the conditions employed in this study.
Suboptimal blood volume QC: The effect of suboptimal blood volume on RNA quality and transcript stability
The effect of blood volume in the Tempus tubes on RNA quality
Blood volume (ml)
Yield total RNA (ug)
RNA Integrity Number (RIN)
A) Adult blood samples (n = 3)*
7.90 ± 1.50
8.20 ± 0.06
2.19 ± 0.02
2.10 ± 0.23
3.38 ± 0.90
8.13 ± 0.27
2.24 ± 0.05
0.91 ± 0.46
2.94 ± 0.91
8.40 ± 0.08
2.12 ± 0.08
1.05 ± 0.53
1.38 ± 0.59
3.53 ± 2.53
2.51 ± 0.08
0.64 ± 0.32
0.55 ± 0.50
1.13 ± 0.13
1.19 ± 0.44
0.38 ± 0.38
0.07 ± 0.03
1.00 ± 0.00
0.77 ± 0.39
0.05 ± 0.05
B) Cord blood samples (n = 2)
79.65 ± 4.76
7.15 ± 0.78
2.15 ± 0.00
2.12 ± 0.00
74.60 ± 1.23
7.50 ± 0.65
2.17 ± 0.00
2.15 ± 0.01
44.42 ± 1.91
9.10 ± 0.00
2.18 ± 0.01
2.09 ± 0.01
33.92 ± 3.13
9.45 ± 0.04
1.98 ± 0.01
1.61 ± 0.02
4.29 ± 0.11
1.03 ± 0.03
1.25 ± 0.03
0.53 ± 0.01
The relative transcript levels for the six genes are shown in Figure 4B and C. We observed blood–volume dependent reduction in the relative transcript levels for the six genes compared with samples with 3 ml blood volume (Figure 4B and C). Samples with volumes less than 1.5 ml for adult blood and less than 1.0 ml for cord blood showed the highest changes in the relative transcript levels for the six genes, compared with the optimal 3 ml samples (Figure 4B and C). The non-normalized raw Cq-values for adult and cord blood samples show a similar trend (Additional file 3A and B). Furthermore, overfilling the Tempus tubes with cord-blood, i.e. more than 3 ml blood per tube, seems to affect the apparent RNA transcript stability, suggesting that the blood–to–RNA stabilizing reagent ratio limit was exceeded (Figure 4C). Our results indicate that the blood–to–RNA stabilizing reagent ratio is very important for blood RNA quality and transcript stability for adult blood samples, whereas the blood volume is less critical for cord blood samples. Hence, collection of 3 ml of blood per Tempus tube will always be the highly recommended optimal blood volume, but this volume is not always possible when blood collection is carried out in a busy maternity unit. For adult blood samples it is advisable to collect 3 ml blood per Tempus, whereas for cord blood samples, minor deviations from the optimal volume per tube may be acceptable.
Our results indicate that blood samples in a large biobank such as MoBa can be used to obtain high-quality RNA suitable for gene expression analysis, provided that special collection tubes are used. The quality of the RNA isolated from such tubes is crucial for successful transcript profiling analyses. Transportation of samples by standard mail apparently affected the RNA quality and RNA transcript stability, but the changes were moderate and the overall RNA quality of the transported samples was good. Some unexplained changes in gene expression changes seem to be associated with suboptimal blood volumes collected in the tubes. Overall, we demonstrate that satisfactory amounts of high-quality RNA can be achieved from samples routinely collected in a large biobank. This is good news for future projects studying association of disease with altered patterns of gene expression.
Blood collection and study design
Whole blood was collected at a maternity unit in Oslo from the umbilical cord of newborns whose mothers had given their informed consent to participate in the MoBa project. Venous blood samples were obtained via phlebotomy from healthy adult donors among the NIPH staff after informed consent. The study design for blood collection is outlined in Figure 1. Samples were collected either into Tempus™ Blood RNA Tubes (space for 3 ml blood) (Applied Biosystems, Foster City, CA) or PAXgene Blood RNA Tubes (space for 2.5 ml blood) (PreAnalytiX, Qiagen, Germany). Samples collected at NIPH (Oslo) were stored for 2 h at room temperature, then at – 20°C for 24 h and finally transferred to – 80°C for long-term storage. Samples shipped by standard mail were then stored at – 20°C for 24 h after arrival at NIPH, and then transferred to – 80°C for long term storage. All samples were handled as recommended by the manufacturers prior to processing.
The adult blood samples collected to study the effects of transportation were shipped to 4 different locations in Norway, with instructions to treat the tubes the same way (temperature, storage) as tubes collected in the local maternity unit, and then to return the tubes to NIPH as it would be normally done with the collected tubes. The four sets were shipped by standard mail to the maternity units in Bærum (~15 km from Oslo), Kristiansand (~ 320 km), Bergen (~ 520 km) and Kirkenes (~ 2000 km). A fifth set of samples was let at room temperature at NIPH (Oslo/lab). The tubes were sent to the hospitals and then returned by the local staff to NIPH, to comprise a worst-case scenario of double the ordinary mail transportation time. Upon arrival at NIPH samples were kept at RT until the last sample arrived and then they were all frozen at the same time. Temperature was monitored during the transportation using small sensors with memory (Tinytag Talk2, Precision Technic Nordic, Rødovre, Denmark). The Regional Ethics Committee and the Data Inspectorate approved the MoBa study, and participants gave their informed consent.
Total RNA from whole blood collected in PAXgene tubes was extracted according to the manufacturer’s instructions and included DNase I treatment (PreAnalytiX, QIAGEN/BD, Hombrechtikon, Switzerland). Total RNA from blood collected in Tempus tubes was extracted using the Tempus™ 6-Port RNA Isolation Kit Protocol on an ABI PRISM® TM 6100 Nucleic Acid PrepStation according to the manufacturer’s instructions and included DNase I treatment and addition of 1X PBS bringing the total volume to 12 ml prior to processing (Applied Biosystems, Foster City, CA).
An aliquot of 5 μl of each extracted total RNA was used for RNA quality control assessments, while the remaining RNA sample was stored at – 80°C until use.
The concentration of extracted total RNA was measured using NanoDrop ND-1000 spectrophotometer (Fisher Scientific, Norway). RNA purity was estimated by examining the OD 260/280 and the OD 260/230 ratios. The RNA integrity was assessed by Agilent 2100 Bioanalyzer using the Eukaryote total RNA 6000 Nano LabChip kit and Eukaryote total RNA Nano assay according to the manufacturer’s instructions (Agilent Technologies, Palo Alto, CA). The RNA integrity numbers (RIN) were calculated using the Agilent 2100 Expert Software (RIN = 1; low RNA quality to RIN = 10; highest RNA quality).
Quantitative real-time PCR measurement
Total RNA (500 ng) from samples was used as template for the synthesis of cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to manufacturer’s protocol. The amplification reactions were carried out in an Eppendorf MasterCycler (Eppendorf, Hamburg, Germany) with the following steps: 10 minutes at 25°C, 2 hours at 37°C and finally, 5 minutes at 85°C. The quality of the cDNA was assessed by the NanoDrop ND-1000 spectrophotometer (Fisher Scientific), and the cDNA was stored at - 20°C until use.
Quantitative real-time PCR (qPCR) was performed in 96-well PCR plates using a 7500Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The cDNA samples were diluted 10-fold and gene-specific transcription levels were determined in a 20 μl reaction volume in triplicates using TaqMan®Fast PCR Universal PCR Master mix following the manufacturer’s protocol. Commercially available primers and probe assays were from Applied Biosystems: IL1B (PN: Hs00174097_m1), IL8 (PN: Hs00174103_m1), FOS (PN: Hs00170630_m1), MYC (PN: Hs00153408_m1), TP53 (PN: Hs00153340_m1), and CDKN1A (PN: Hs00355782_m1), and 18S rRNA (PN: Hs99999901_s1). The PCR cycling program included an enzyme activation step at 95°C for 20 seconds, and then 40 cycles of annealing and extension steps at 95°C for 3 seconds and 60°C for 30 seconds, respectively.
The quantification cycle (Cq; standard name for Ct and Cp ) values were recorded with SDS v1.3 software (Applied Biosystems, Foster City, CA); the Cq value is the fractional cycle number at which the fluorescence exceeds a fixed threshold. The raw Cq-values were then exported into Excel- files and analyzed by the comparative Cq – method [17, 18] using 18S rRNA as internal control. Targets with Cq-values > 35 were considered beyond the limit of detection (LOD) and all Cq-values above 35 were coded as missing values. For downstream analysis missing values were replaced by Cq (LOD) + 1 (i.e., 35 + 1: Cq = 36). Target genes were normalized to 18S rRNA internal controls, [this is given by ΔCq; where ΔCq (sample) = Cq (target gene) – Cq (internal controls)]. The ΔΔCq values were generated by subtracting the ΔCq-value for the reference samples (calibrators) from the ΔCq-value for the samples [ΔΔCq = ΔCq (sample) – ΔCq (calibrator); fold change = 2-ΔΔCq. A reference sample was determined within each study, and for samples without an obvious reference sample (calibrator), the linear scale of ΔCq (sample); 2-ΔCq was used [17, 18]. The fold change values were then log2-transformed in order to make the values symmetrical around zero. The 18S rRNA stability was evaluated and results are presented in Additional file 4. These four genes: CDKN1A, IL8, MYC, and TP53 were analyzed when the two RNA stabilization systems (PAXgene and Tempus) were compared, and then two more genes (FOS and ILB) were added, i.e. six genes were analyzed in the remaining study.
Statistical analysis of RNA yield, purity, integrity and ΔCq - values was carried out by one-way analysis of variance (ANOVA), followed by post hoc Dunnett’s tests to allow for multiple comparisons or by non-parametric Kruskal-Wallis test. Normal distribution and equality of variances was evaluated using the Shapiro-Wilk test and the Levene's test of homogeneity of variance. Two-sample t-test was used to compare cord blood samples collected in the PAXgene vs the Tempus tubes. The data are presented as means ± SE. Statistical analyses were performed using SPSS v17 software (SPSS, Inc., Chicago, IL), and p < 0.05 was accepted as statistically significant.
Quantification cycle Threshold
Quantitative Real-time PCR
RNA Integrity Number
The Norwegian Mother and Child Cohort Study
Norwegian Institute of Public Health
This study was supported by ABC (US National Institutes of Health award NS047537) and NewGeneris (Contract No 016320–2). MoBa is supported by the Norwegian Ministry of Health, the Ministry of Education and Research, NIH/NIEHS (contract no. NO1-ES-85433), NIH/NINDS (grant no.1 UO1 NS 047537–01), and the Norwegian Research Council /FUGE (grant no. 151918/S10). We thank Jorid Eide for help with collection of the cord blood specimens.
We also thank Applied Biosystems in Norway and Sweden (Tempus™) and VWR Norway (PAXgene™) for supplying blood RNA stabilizing collection tubes and associated kits during the comparison of the two blood RNA stabilizing systems study. VWR Norway was PAXgene™ Blood RNA system distributor in Norway at that time.
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