MicroRNAs (miRNAs) are small non-coding RNA molecules that play important roles in a variety of normal and disease-related biological processes by post-transcriptionally regulating the expression of target mRNAs [1,2]. They have been detected in many body fluids, because circulating or extracellular miRNAs that bind to protein complexes or are contained within membranous particles such as exosomes or microvesicles are protected from degradation by RNase [3-7]. Therefore, plasma miRNA molecules are stable and a potential source of novel biomarker for a number of diseases, including cancer [8,9].

However, the amount of plasma miRNA molecules is known to be markedly affected by sample collection or storage methods. For example, hemolysis, which is caused by negative pressure during sample extraction, results in reddish color tone and marked increase in the amounts of plasma miRNA, whereas the freeze-thaw process decreases the amounts. Thus, although circulating miRNAs are useful biomarkers for many diseases, objective quality checks on plasma samples should be performed carefully.

In the present study, a potential objective method for quality checking plasma samples was examined using the macroscopic color tone and absorbance. And the optimal absorbance cut-off values for a quality check of plasma samples were validated using the amounts of plasma miRNA molecules.

Clinical samples

A total of 1213 plasma samples were collected from patients who had been diagnosed with digestive cancer and underwent surgery between January 2012 and April 2015 at Kyoto Prefectural University of Medicine. Written informed consent was obtained from all patients.Control samples were also collected from 10 adult healthy volunteers by standard cubital vein puncture.

Storage of plasma samples

Immediately after the collection of blood samples in sodium heparin tubes (BD Vacutainer, Franklin Lakes, NJ, USA), cell-free nucleic acids were isolated using a three-spin protocol (1500 rpm for 30 min, 3000 rpm for 5 min, and 4500 rpm for 5 min) in order to prevent contamination by cells or cellular nucleic acids. 120 μl of each plasma sample was added to the well of a 96-well plate and stored at -80°C until the absorbance was measured. The remainder of each plasma sample was stored at -80°C until further processing.

RNA extraction

Total RNA was extracted from 400 μl of plasma samples using a miRVana PARIS kit (Ambion, Austin, TX, USA) and eluted into 100 μl of pre-heated (95°C) elution solution according to the manufacturer’s instructions. After plasma samples were thawed on ice and treated with Qiazol, synthetic Caenorhabditis elegans miRNA oligonucleotides, celmiR- 39 (a mixture of 25 fmol of each oligonucleotide in a total volume of 5 μl) were added to each plasma sample in order to normalize the efficacy of RNA extraction. RNA samples were stored at -80°C until further processing.

Study design

The study design is summarized in Figure 1. This study was divided into five steps: (1) Determination of the permitted color tone using intentionally hemolyzed plasma samples; i.e., to determine whether the samples were reddish or not, and (2) Examination of absorbance at 414 nm of the clinical plasma samples. (3) Decision of the optimal cut-off absorbance value for a quality check. (4) miRNA microarray analysis between fresh and intentionally hemolyzed plasma samples from a healthy volunteer, in order to select candidate miRNAs for validation. (5) Validation study of the optimal cut-off absorbance value in clinical plasma samples using the amounts of candidate miRNAs.

Figure 1: Study design.

Measurement of absorbance in plasma samples

The absorbance values of plasma samples were measured using a fluorescent microplate reader with XFLUOR4 (TECAN, Mannedorf, Switzerland) at a wavelength of 414 nm (reference wavelength: 595 nm) according to the previous study [10].

MicroRNA microarray analysis

Plasma samples from healthy volunteers were analyzed by a microRNA microarray. The fresh sample and intentionally hemolyzed one due to negative pressure were examined [11-13]. Microarray analyses were performed using the 3D-Gene microRNA microarray platform (TORAY, Kamakura, Japan) [14,15], and RNA extraction was conducted according to the manufacturer’s instructions. Only a very small amount of total RNA was present in the plasma samples; therefore 2 out of the 4 μl of total RNA extracted from 300 μl of plasma samples were used in microarray experiments. RNA was labe15led with Hy5 and hybridized at 32°C for 16 h on the 3D-Gene chip. The 3D-Gene miRNA microarray contained more than 1000 miRNA molecules based on the Human miRNA Version 15 of MirBase (http://microrna.sanger.ac.uk/). We analyzed microarray analysis data and selected three candidate miRNA molecules, the levels of which were significantly higher in hemolyzed samples than in fresh samples or similar between both samples.

MicroRNA detection protocol

The amounts of target miRNA molecules in plasma were measured by RT-PCR. A reverse transcription reaction was carried out using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), and the amount of each miRNA molecule was quantified in duplicate by quantitative RT-PCR (qRT-PCR) with the human TaqMan MicroRNA Assay Kit (Applied Biosystems) in accordance with previously described protocols [16]. These assays were run on a Step One Plus PCR system v2.1 (Applied Biosystems). The primers for human miRNA (i.e., hsa-miR-16, hsa-miR-19b, and hsamiR- 223) and C. elegans miRNA (i.e., cel-miR 39) used in the TaqMan assays were obtained from Applied Biosystems. Changes in the plasma miRNA levels were calculated using the 2-ΔΔCt method [17,18].

Statistical analysis

The paired t-test was used to compare the results obtained for fresh and hemolyzed plasma samples. P-values of <0.05 were considered significant. ROC curves and the AUC were used to assess the most appropriate absorbance cut-off value for detecting hemolyzed plasma samples, which was determined with the Youden index [19]. Spearman’s correlation co-efficient was used to investigate the relationship between absorbance and miRNA levels.

Examination of the permitted color tone and absorbance in control sample

The color series of intentionally hemolyzed plasma samples were shown in Figure 2a. The macroscopic color tone gradually became dense, and the permitted color tone as the not-hemolyzed plasma sample was determined. A borderline of absorbance value at 414 nm, whether the samples were hemolyzed or not, was between 1.363 and 1.761.

Figure 2: Evaluation of the most appropriate absorbance cut-off value for detecting damaged plasma sample. (a) Determination of the permitted color tone level using intentionally hemolyzed plasma samples. (b) Evaluation of the optimal plasma absorbance cut-off value in 1213 clinical plasma samples. Absorbance at 414 nm and the macroscopic color tone of the plasma samples were assessed. An analysis was performed based on these data, and the Youden index was used to determine the optimal absorbance cut-off value for detecting hemolyzed samples.

Evaluation of the most appropriate absorbance cut-off value for detecting damaged plasma sample

Absorbance value at 414 nm and macroscopic color tone were assessed in 1213 plasma samples. The mean and standard deviation of absorbance value were 1.086 and 0.662. A receiver operating characteristic (ROC) analysis of absorbance value and color tone of each sample revealed the optimal absorbance cut-off value for detecting damaged plasma sample. The area under the ROC curve (AUC) was very high (AUC=0.986) at an absorbance cut-off value of 1.664 (Figure 2b).

Results of the miRNA microarray analysis and validation study

Table 1 shows a list of miRNA molecules whose levels were higher in hemolyzed samples than in fresh samples, or similar in both samples. The miRNAs that were not detectable in fresh samples were not listed in Table 1. Three miRNA molecules, miR-16, miR-19b, and miR-223, were selected for the validation study of this quality checking method, because these miRNAs were present at high amounts in plasma and markedly different between hemolyzed and normal samples (Table 1, squarish marks).

Table 1: Results of the miRNA microarray analysis. MicroRNA molecules whose concentrations were increased or unchanged in hemolyzed plasma samples.

The amounts of miR-16 and miR-19b were significantly increased with approximately 100- and 60-times in hemolyzed plasma samples, respectively (p<0.0001 in both cases; paired t-test; Figures 3a and 3b), whereas that of miR-223 remained unchanged in fresh and hemolyzed samples (p=0.169, Figure 3c). The absorbance of these plasma samples at 414 nm was also examined, and it was found that the absorbance value correlated with the amounts of miR-16 or miR-19b (p<0.001; Spearman’s correlation co-efficient; Figure 3d, data for only miR-16 was shown).

Figure 3: Validation study of paired fresh and hemolyzed plasma samples. The amounts of plasma miR-16, miR-19b, and miR-223 were examined by RT-PCR (a, b, and c). Differences between fresh and hemolyzed samples were analyzed using the paired t-test. NC: fresh samples, NC H: hemolyzed samples. (d) The relationship between 414 nm absorbance value and the amount of plasma miR-16 was analyzed in intentionally hemolyzed samples obtained from healthy volunteers.

Absorbance values and miRNA amounts in clinical plasma samples

The absorbance values and amounts of plasma miR-16 and miR- 19b were examined in 10 clinical plasma samples obtained from gastric or pancreatic cancer patients (Table 2). Tumor progression of these 10 randomly selected patients was various degree. The samples obtained from 5 patients were macroscopically reddish, and the others were not. The absorbance values at 414 nm of 5 reddish samples were more than 1.664, while those of the others were less than 1.664. The amounts of miR-16 and miR-19b were also increased in the samples with higher absorbance values regardless of their tumor progression (p=0.0001 and p=0.0003 for miR-16 and miR-19b, respectively; Figures 4a and 4b), however no significant difference was observed in the amounts of miR- 223 between both groups (p=0.241, data was not shown).

Table 2: Clinical features and absorbance values of clinical plasma samples. These ten random samples exhibited various degree of tumor progression. Five samples were macroscopically reddish, and the other five were not.

Figure 4: Comparison of plasma miRNA levels in cancer patients according to absorbance value. Plasma miR-16 (a) and miR-19b (b) levels of cancer patients are shown relative to the level of PK1. PK1, PK2, MK1, MK2, and MK3 exhibited absorbance values of less than 1.664 at 414 nm. PK3, PK4, MK4, MK5, and MK6 exhibited absorbance values of more than 1.664 at 414 nm.

The quality of clinical sample is extremely important when researching new plasma biomarkers. Although many studies identified plasma miRNAs as disease biomarker and previous studies reported a relationship between hemolysis and the increase of plasma free nucleic acid [10], only a few studies have examined the effects of hemolysis and other damaging processes on clinical plasma researches. We also previously identified some plasma miRNA biomarkers, however only macroscopic color tone was mainly checked for the quality of clinical plasma samples. In the present study, we confirmed a more objective method for quality checking plasma samples based on their color tone and absorbance values.

During hemolysis, the cell membranes of red corpuscles are damaged physically, chemically, or biologically by various factors, and their protoplasm leaks into the bloodstream. Thus, when clinical blood samples are extracted, clinicians need to be aware that surplus negative pressure may cause hemolysis. Michaela et al. reported that plasma miRNA concentration was affected by hemolysis, and the degree of hemolysis in a particular plasma sample was able to be determined from its macroscopic color tone or absorbance at 414 nm [10]. Colin CP et al. also showed that blood cells were a major contributing factor to circulating miRNA levels and that blood cell count or hemolysis markedly altered plasma miRNA levels. Therefore, plasma miRNAbased data should be carefully interpreted, because the majority of the reported plasma miRNA biomarkers of cancer are strongly expressed in blood cells. The change of a particular miRNA biomarker may reflect the blood cell-based changes rather than cancer-specific ones [11,12]. Thus, clinical samples need to be handled very carefully when blood cellderived miRNA molecules are examined as plasma biomarkers [13].

Some difficulties were associated with quality checking plasma samples. Chyle-containing samples were sometimes found, and the absorbance of these samples was slightly higher than that without hemolysis or chyle (data not shown). The chyle contained in plasma samples originates from various sources including chylomicrons,neutral fats, very low-density lipids, and intravenously administered fat tablets. Further studies are needed to confirm whether these samples are suitable for biomarker-based experiments. On the other hand, cancer and other diseases may affect the amounts of plasma miRNAs. If cancer-specific miRNAs are used to quality check of plasma samples, we may inappropriately exclude suitable samples and miss the cancerspecific changes. Therefore, the candidate miRNA molecules used for quality checking should be selected carefully, and some miRNAs should be examined repeatedly.

In the present study, the samples which showed higher absorbance more than the cut-off value contained significantly larger amounts of miR-16 and miR-19b than those showed lower absorbance value. These results may indicate that the amounts of these miRNAs are influenced by hemolysis rather than tumor progression, and support the accuracy and reproducibility of this cut-off absorbance value. The measurement of absorbance is an easy and objective method for quality checking plasma samples. Although plasma samples are very useful for a range of biomarker analyses, it may be better to discard samples with higher absorbance.

A simple method for quality checking plasma samples using color tone and absorbance is very useful and significant.

None of the authors have any conflict of interest to disclose.