The Effect of Blood Clots on the Quality of RNA Extracted from PAXgene Blood RNA Tubes

Abstract

Background:

PAXgene^® Blood RNA tubes are routinely used in clinical research and molecular biology applications to preserve the stability of RNA in whole blood. However, in practice, blood clots are occasionally observed after blood collection and are often ignored. Currently, there are few studies on whether blood clots affect the quality of RNA extracted from these tubes.

Materials and Methods:

Fifteen pairs of non-clot and clot PAXgene Blood RNA tube samples (n = 30) were collected to form two matched groups from 15 patients. According to the maximum diameter (d) of the blood clot observed visually at the time of sample reception, the clot groups were divided into a small-clot group (0 cm < d < 0.5 cm) and a large-clot group (d ≥ 0.5 cm). RNA was extracted by the PAXgene Blood RNA Kit. To analyze the quality of RNA, its yield and purity were assessed by spectrophotometry, and integrity was measured by microfluidic electrophoresis. An A_260/280 ratio between 1.8 and 2.2 indicated purified RNA, and RNA integrity number (RIN) values ≥7.0 were considered to represent qualified integrity.

Results:

The median yields of RNA from the non-clot and clot groups were 3.84 (2.80–6.38) μg and 4.87 (2.77–8.30) μg, respectively. The median A_260/280 ratios were 2.08 (2.06–2.09) and 2.09 (2.07–2.11), whereas the median A_260/230 ratios were 1.77 (1.31–1.91) and 1.67 (1.21–1.94) in the two groups. In addition, the median RINs were 8.20 (8.00–8.40) and 7.20 (6.60–7.70), respectively. There were no significant differences in RNA yields, A_260/280, or A_260/230 between the two groups. However, the RIN value of the clot group was significantly lower compared with the non-clot group (p < 0.05), with RIN ≥7.0 found in all non-clot samples and 60% of clot samples (p < 0.05). Furthermore, in the clot groups, the small-clot samples had higher RIN values than large-clot samples (8.25 [7.75–8.75] vs. 6.90 [6.60–7.30], p < 0.001).

Conclusions:

The formation of large blood clots in PAXgene Blood RNA tubes will reduce the integrity of extracted RNA.

Background

Biological samples provide valuable clinical resources for translational medicine research. Among them, blood samples are widely utilized in the fields of pathogenic mechanisms, immune regulation, drug development, etc. During the “preanalytical” stage, without proper handling, the composition of blood samples may change significantly in the processes of collection, storage, and transportation, resulting in unpredictable errors in subsequent scientific research and clinical applications.¹

Gene expression profiling in peripheral blood is frequently performed to identify susceptibility genes or biomarkers for human traits or diseases.² Downstream RNA analyses include quantitative real-time reverse transcription‒PCR, RNA sequencing, northern blot analysis, RNA mapping, and microarray analysis. The accuracy of gene expression evaluation is recognized to be influenced by the quantity and quality of RNA.³ However, RNA is prone to degradation during the collection process.⁴ Since blood RNA is highly sensitive to enzymatic degradation by ubiquitous ribonucleases, many environmental factors reduce RNA quality.⁵ At the same time, RNA is chemically unstable and susceptible to self-hydrolysis through transesterification reactions catalyzed by temperature, alkaline pH, and water.⁶ Therefore, PAXgene^® Blood RNA tubes (PreAnalytiX, Qiagen, Germany), which allow instant preservation of RNA, are widely used.⁷ The RNA collection system uses proprietary reagents to inhibit RNA degradation in blood cells immediately after collection.⁸

Preanalytical variables in the collection of blood samples, if not controlled and recognized correctly, may impact sample quality and, consequently, the accuracy of molecular analysis.⁹ During sample collection, blood clots of varying sizes were occasionally found in the PAXgene Blood RNA tubes. Previous studies have investigated the effects of storage time, temperature, and repeated freeze‒thaw cycles on RNA extracted from PAXgene Blood RNA tubes, but little has been explored regarding blood clots.^10,11 In this study, we aimed to evaluate the RNA yield, purity, and integrity of clot samples compared with matched non-clot samples and to determine the potential factors that may influence the quality of RNA extracted from PAXgene RNA tubes.

Materials and Methods

Sample collection and study design

From December 2019 to April 2020, our biobank received a total of 15 PAXgene Blood RNA tube a samples containing blood clots. The biobank technician immediately informed the nurse to recollect a blood sample from the same patient within 2 days, and confirmed that the newly collected sample was free of blood clots. Paired sets of samples were stored at −80°C until extraction and detection. Then we investigated the effect of clots on the quality of RNA extracted from PAXgene blood RNA tubes; the 15 paired samples included both the non-clot and clot groups. All samples received were examined by the same technician to determine the presence of blood clots. According to the maximum clot diameter observed visually at the time of sample reception, blood clots were divided into small clot (0 < d < 0.5 cm, n = 4) and large-clot (d ≥ 0.5 cm, n = 11) samples. Each tube contained 2.5 mL of whole blood and 6.9 mL of additives.¹² Fasting venous blood samples were collected from patients in the early morning after obtaining informed consent.

RNA extraction

Before RNA extraction, frozen PAXgene samples were thawed at room temperature for 2 hours and then inverted 10 times. Manual extraction of RNA from the two paired groups was performed using the PAXgene Blood RNA Kit (#762174; QIAGEN) according to the manufacturer's instructions with DNase treatment. RNA was eluted in 70 μL elution buffer in 1.5-mL tubes, kept on ice, and analyzed immediately.

Quality analysis of RNA

The quantity and purity of RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA yield was measured by absorbance at 260 nm, and RNA purity was estimated by examining the OD 260/280 and OD 260/230 ratios.¹³ An A_260/280 ratio between 1.8 and 2.2 indicates highly purified RNA with minimum DNA contamination.¹⁴

The RNA integrity was assessed by an Agilent 2100 Bioanalyzer with 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, USA). In addition, RNA integrity numbers (RIN) were calculated using Agilent 2100 Expert Software.¹⁵ The RIN ranges from 1 to 10. An RIN value of 10 indicates high RNA integrity, while a value of 1 indicates severe RNA degradation. In previous studies, RIN values ≥7.0 were considered ideal for high-throughput downstream applications.^16,17 In our experiment, RNA integrity was considered acceptable when RIN ≥7.0.

Institutional Review Board statement

Protocols for obtaining blood samples from patients were reviewed and approved by the Ethics Committee of Jinling Hospital (2017NZKY-013-06). All experiments were performed in compliance with the Declaration of Helsinki.

Informed consent statement

Written informed consent was obtained from all participants involved in the study.

Statistical analysis

Statistical analysis was performed using IBM SPSS 20.0 software, and p < 0.05 was considered statistically significant. Normal distribution was tested using the Shapiro‒Wilk test. To compare the difference between the non-clot and clot groups, the paired sample Wilcoxon signed-rank test was used for analysis. Fisher's exact test was used for differential analysis of count data. In addition, differences in yields, A_260/280, A_260/230, and RIN values among varying clot size groups were analyzed by Mann–Whitney U test.

Results

In this study, paired samples were collected within 2 days. A total of 30 PAXgene blood RNA tube samples from 15 patients were stored at −80°C for 1–5 months before RNA extraction. After thawing, blood clots were not observed in the non-clot group, whereas samples from the clot group still had clots in the tubes. All blood clots were included for RNA extraction.

The median RNA yields from the non-clot group and clot group were 3.84 (2.80–6.38) μg and 4.87 (2.77–8.30) μg, respectively (Fig. 1). We assessed the RNA purity by measuring the A_260/280 and A_260/230 ratios. The median values of A_260/280 were 2.08 (2.06–2.09) and 2.09 (2.07–2.11) (Fig. 2). The A_260/280 of all samples was between 2.04 and 2.14. The median values of A_260/230 were 1.77 (1.31–1.91) and 1.67 (1.21–1.94), respectively (Fig. 3). There were no significant differences in yield, A_260/280 or A_260/230 between the non-clot and clot groups.

FIG. 1.

The median yields of RNA extracted from two paired groups. The yields of RNA extracted from the PAXgene blood RNA tubes of the non-clot group (n = 15) and clot group (n = 15). There were no significant differences in RNA yields between them.

FIG. 2.

The median A_260/280 values of RNA extracted from two paired groups. No significant differences were found in A_260/280 ratios between the non-clot (n = 15) and clot (n = 15) groups.

FIG. 3.

The median A_260/230 values of RNA extracted from two paired groups. No significant differences were found in A_260/230 ratios between the non-clot (n = 15) and clot (n = 15) groups.

At the same time, median RIN values were recorded as 8.20 (8.00–8.40) and 7.20 (6.60–7.70) in the non-clot and clot groups, respectively. The RIN value of the clot group was significantly lower compared with the non-clot group (p < 0.05). RIN ≥7.0 was found in all samples of the non-clot group. In the clot group, 9 samples had RIN ≥7.0, accounting for 60%. Since the purity of all samples in the non-clot and clot groups met the target based on the RIN value (≥7.0), all samples in the non-clot group were deemed of acceptable quality, whereas only 60% of samples in the clot group met the same standard. The qualified rate was higher in the non-clot group compared with the clot group (p < 0.05, Table 1).

Table 1.

RNA Integrity Number Values and Qualified Rates of the Non-clot and Clot Groups

Group	RIN value	RIN value ≥7.0, n (%)
Non-clot group (n = 15)	8.20 (8.00–8.40)	15 (100%)
Clot group (n = 15)	7.20 (6.60–7.70)	9 (60%)
p	<0.05	<0.05

RIN, RNA integrity number.

To study the influence of different sizes of blood clots on RNA quality, the 15 PAXgene Blood RNA tube samples in the clot group were divided into the small-clot group (n = 4) and the large-clot group (n = 11) according to the clot size observed visually at the time of sample collection. No significant difference was found in yield, A_260/280, or A_260/230 between small- and large-clot samples. The RIN values of the two groups were 8.25 (7.75–8.75) and 6.90 (6.60–7.30), respectively. The RIN values of the small-clot group were significantly higher than those of the large-clot group (p < 0.001). In addition, the RIN values of all samples in the small-clot group were ≥7.0, whereas only 45% of samples in the large-clot group had RIN values ≥7.0 (Table 2). Therefore, we conclude that blood clots, especially those with diameters ≥0.5 cm, will affect the integrity of RNA extracted from PAXgene Blood RNA tubes.

Table 2.

RNA Integrity Number Values and Qualified Rates of the Small-Clot and Large-Clot Groups

Group	RIN value	RIN value ≥7.0, n (%)
Small-clot group (n = 4)	8.25 (7.75–8.75)	4 (100%)
Large-clot group (n = 11)	6.90 (6.60–7.30)	5 (45%)
p	<0.001	>0.05

Discussion

During routine collection of PAXgene Blood RNA tube samples, the occasional presence of blood clots in the tube can be easily ignored. There are few literature reports about the effect of blood clots on the quality of RNA extracted from tubes. In one of our previous studies, 19 samples were observed with blood clots among 300 samples of PAXgene Blood RNA tubes. The RNA yield and integrity of blood clot samples were significantly lower than those of non-clot samples.¹⁸ However, only one PAXgene tube was collected from every patient at one time point. Therefore, it was impossible to detect the quality of samples from individual patients. Instead, samples from different patients at the same time were selected for comparison.

In our current study, non-clot and clot samples from each patient were paired to investigate the effect of clots on the quality of RNA extracted from PAXgene blood RNA tubes, ensuring comparability of the research data. To ensure consistency between different sample operations, all PAXgene blood RNA tube samples were stored at −80°C and then RNA extraction and quality tests were performed by the same technician using the same batch of reagents at the same time.

If blood is not handled properly upon sampling, it will lead to RNA degradation. For example, after collection, insufficient mixing of tubes with additives led to the formation of microclots, which would adversely influence RNA extraction. In addition, after long-term storage, blood clots in the tubes might increase and affect the quality of RNA. Preanalytical variables play a crucial role in RNA stability.¹⁹ In a study on the preanalytical robustness of blood collection tubes, the integrity of RNA extracted from PAXgene blood tubes was found to be sensitive to effective inversion and storage temperature.²⁰

In this study, we focus on the effect of blood clots on the quality of RNA extracted from PAXgene blood RNA tubes: a significant reduction in RNA integrity was observed in samples with blood clots, especially those with large clots. In our study, all subjects had normal blood coagulation tests. Therefore, we speculated that several factors, such as the failure to invert and gently mix the tube in time during blood collection, insufficient temporary storage time before transferring to low temperature containers, or improper collection sequence of the PAXgene tube, might lead to the formation of blood clots. Environmental factors, such as low temperatures or high pressure, may also cause blood clots to form after blood drawing (data not published). It is increasingly recognized that preanalytical factors, if not properly recognized and controlled, can affect sample quality and subsequent molecular analysis.⁹ Therefore, the establishment of robust and standardized protocols is a prerequisite to minimize the impact of possible factors on sample quality and stability. This is particularly important to collect blood samples for large-scale biobanks.¹⁵

Considering the huge impact of the preanalytical phase on sample quality, sample collection and preprocessing must be performed using reproducible standard operation procedure to minimize intersample variability. According to the collection instructions of the PAXgene Blood RNA tube (www.PreAnalytiX.com), there are some precautions when drawing blood. First, sample collection staff should ensure that the PAXgene Blood RNA tube is kept at room temperature (18°C–25°C) before use. If the PAXgene tube is the only tube to be drawn, a small amount of blood should be drawn into a “discard tube” before drawing blood into the sample tube. Otherwise, the PAXgene tube should be the last tube drawn in the phlebotomy procedure. During blood collection, the tube was held vertically below the blood donor's arm. Immediately after blood collection, the tube was gently inverted 8–10 times. Finally, the samples were stored upright at room temperature for a minimum of 2 hours and a maximum of 72 hours before processing or transferring to a refrigerator (2°C–8°C) or freezer (−20°C or −80°C).

In other studies, RNA integrity was influenced by increased storage temperature.¹⁰ It has been well documented that RNA molecules are sensitive to physical degradation due to high temperature.²¹ It is important for biobanks and laboratories to carefully validate the robustness of their processing methods for blood collection to avoid the effects of preanalytical variables.²²

In conclusion, large blood clots reduce the integrity of RNA extracted from PAXgene blood RNA tubes. Therefore, samples should be collected strictly according to standard procedures and examined carefully when received.

Footnotes

Acknowledgments

The authors would like to thank the patients for their contributions to this study. All patient samples were obtained from the Renal Biobank of the National Clinical Research Center of Kidney Diseases, Renal Biobank of Jiangsu Provincial Science and Technology Resources Coordination Service Platform.

Authors' Contributions

C.Z. designed the study and reviewed the article. R.T. performed the experiments, analyzed the data, and wrote the article. L.Z. modified the article and made the figures. P.Z. and R.Y. were responsible for resources. All authors have read and agreed to the final version of the article.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was supported by the Natural Science Foundation of Jiangsu Province (BK20221552) and the Key Project of Military Health Care (22BJZ43).

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