|Year : 2021 | Volume
| Issue : 4 | Page : 186-192
Achieving the best RNA quality in urologic tumor samples intended for transcriptome analysis
Tzu-Chun Lai1, Tai-Lung Cha2, Yi-Ta Tsai2, Shu-Yu Liu1, Sheng-Tang Wu1, En Meng1, Chih-Wei Tsao1, Chien-Chang Kao2, Chin-Li Chen1, Guang-Huan Sun1, Dah-Shyong Yu1, Ming-Hsin Yang2
1 Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
2 Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center; Graduate School of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
|Date of Submission||16-Apr-2021|
|Date of Decision||23-Jun-2021|
|Date of Acceptance||07-Jul-2021|
|Date of Web Publication||14-Dec-2021|
Dr. Ming-Hsin Yang
Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, No. 325, Section 2, Chenggong Road, Neihu District, Taipei 114
Source of Support: None, Conflict of Interest: None
Purpose: To conduct research on the molecular oncology, physiology, and immunology of urologic tumors requires dissociated viable samples. Improper collection compromises the quality of data attained in molecular and functional assays due to the increased quantities of degraded proteins and RNA. We sought to improve the methods for tissue collection which can avoid generating considerable loss in the viability of cells for further analyses. Materials and Methods: Fifty resected tumor samples from 35 patients were obtained with different surgical techniques and at various time points for viability and RNA quality evaluation. The degradation of RNA was evaluated by its Qubit IQ score, OD 260/280 ratio, total yield, and quantity of β-actin. Results: Snap-frozen tissue samples obtained within 30 min showed better cell viability (P < 0.0001), RNA total yield (P = 0.0081), Qubit ratio (P = 0.003), OD 260/280 ratio (P = 0.4213), and quantity of β-actin (P = 0.0015). Moreover, the bladder tumor samples collected from transurethral biopsy presented more satisfied cell viability results than the ones resected by transurethral electroresection (P < 0.0001). Conclusion: Tumor samples should be processed or frozen freshly within 30 min once removed from human body. Furthermore, transurethral biopsy of bladder tumor is considered a better method for collecting samples for further molecular oncology studies. The high-quality RNA produced enable researchers to conduct more reliable studies by avoiding the experimental artifacts due to the presence of cellular debris or dead cells.
Keywords: RNA quality, transurethral biopsy, transurethral electroresection, urologic tumor
|How to cite this article:|
Lai TC, Cha TL, Tsai YT, Liu SY, Wu ST, Meng E, Tsao CW, Kao CC, Chen CL, Sun GH, Yu DS, Yang MH. Achieving the best RNA quality in urologic tumor samples intended for transcriptome analysis. Urol Sci 2021;32:186-92
|How to cite this URL:|
Lai TC, Cha TL, Tsai YT, Liu SY, Wu ST, Meng E, Tsao CW, Kao CC, Chen CL, Sun GH, Yu DS, Yang MH. Achieving the best RNA quality in urologic tumor samples intended for transcriptome analysis. Urol Sci [serial online] 2021 [cited 2022 May 21];32:186-92. Available from: https://www.e-urol-sci.com/text.asp?2021/32/4/186/332411
| Introduction|| |
It is necessary to process, store, archive, and distribute human tumor samples and their related clinical records, which are used to develop understanding of biomedical characteristics, metastatic potential, and sensitivity to subsequent therapy. The quality of RNA samples, which is paramount to any downstream application involving this nucleic acid, also relies on the viability of tumor samples. Well-collected and properly preserved frozen tissues are ideal for conducting transcriptomic, genomic, and proteomic research.,, Since factors such as nucleic acid degradation or a lack of purity may have a significant effect on the results of studies, the quality of the samples collected from patients must be taken into consideration when molecular analyses of nucleic acids are being conducted.,,, Therefore, as quality control, it is crucial to ensure the appropriateness of tumor tissues available for molecular analyses used in modern translational research.,,,,,, In this regard, it is important to check the RNA integrity and purity of obtained samples.,,,,,,, For RNA samples, the purity can be assessed by the ratio of absorbance at 260 and 280 nm. RNA sample integrity can be observed by agarose gel or microchip electrophoresis, the RNA integrity number (RIN) can be calculated, and genes can be amplified by real-time polymerase chain reaction (RT-PCR). Furthermore, RIN can be evaluated using two unique dyes: one that selectively binds to highly structured RNA and one that binds to small degraded RNA. Thus, in the present study, we collected renal cell carcinoma (RCC) and urothelial carcinoma (UC) samples during surgeries at different time points for purpose of viability and RNA sample quality analyses. It is also possible that tumor resection techniques could influence sample quality. For UC in the urinary bladder, transurethral electroresection is the most common procedure for resection of the tumor lesion; however, this technique might adversely affect cell viability and RNA quality. Hence, we also collected samples using both transurethral biopsy [[Figure 1]a, right panel] and transurethral electroresection [[Figure 1]a, left panel] to evaluate differences between samples collected via different methods.
|Figure 1: (a) Representative images of transurethral electroresection and transurethral biopsy. (b) Representative image of flow cytometry Annexin-V/PI analysis for cancer cells. Left, fresh tumor processed within 30 min. Right, fresh tumor processed at 60 min|
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| Materials and Methods|| |
Patients and tumor sample collection
All patients signed their informed consent to our study, which was approved by our Ethical Committee (TSGHIRB NO: 2-107-05-167). The study was conducted in accordance with the guidelines stipulated in the Helsinki Declaration. The study included 35 cancer patients treated at Tri-Service General Hospital, Taipei, Taiwan. For each patient, tumor samples were collected during surgery and sent to the laboratory on ice within 30 min. Samples were divided into several pieces and processed or snap frozen in liquid nitrogen at 30, 45, and 60 min after collection. The snap-frozen samples were then processed 1 month after the collection day.
Using scalpels, primary human tumors were crosscut into small pieces and minced completely until nearly liquid. Tissues were incubated in Dulbecco's modified Eagle's medium/F12 (Gibco, Grand Island, NY, USA) with 3 mg/mL collagenase type II (Sigma-Aldrich, St. Louis, MO, USA) and 1% fetal bovine serum at 37°C for 2 h to digest tissue. After incubation, the dissociated tissue was mixed vigorously by pipetting for 1 min and was then passed through a 70-μm cell strainer (Corning, New York, USA). The cell pellet was subsequently centrifuged at 1500 rpm for 5 min and washed with phosphate-buffered saline (PBS) twice.
Flow cytometry and cell viability analysis
Viability was assessed using an Annexin-V Apoptosis Detection Kit with propidium iodide (BioLegend, San Diego, CA) following the tailored protocol provided by supplier. Briefly, dissociated cells obtained from tumor digestions were harvested using 1% trypsin-ethylenediaminetetraacetic acid, centrifuged at 1500 rpm for 5 min, and then washed with PBS. Cells were then blocked in 1 ml of flebogamma 5% (Grifols Biologics) at 4°C for 30 min. After incubation, cells were centrifuged at 1500 rpm for 5 min in preparation for staining. Cells were then stained with FITC-Annexin-V and propidium iodide at room temperature for 15 min. Samples were subsequently analyzed by flow cytometry using BD FACS Fortessa with appropriate filters. Data were then analyzed with FlowJo 10.0 software (Tree Star).
RNA extraction and analysis
To disrupt the cells, they were washed three times in PBS and 350-μl Buffer RLT lysis buffer (Qiagen). RNA was then extracted according to the protocols from the RNeasy Mini Handbook (Qiagen). Purity and quantity were analyzed using the ND1000 Spectrophotometer with absorbance read at 260/280 nm (NanoDrop, Wilmington, US). All samples were expected to have a 260/280 ratio of approximately 2, indicating that the RNA was pure and free of contaminants. RNA quality was also examined using a Qubit RNA IQ Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit 4 Fluorometer (Thermo Fisher Scientific).
Complementary DNA synthesis and quantitative real-time polymerase chain reaction
Complementary DNA (cDNA) was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). For 1 μg of RNA, 2 μl of DNAse was added per sample for DNA wipeout following incubation for 5 min at 42°C on an Eppendorf Mastercycler Thermal Cycler (Eppendorf, UK). Reverse Transcriptase (RT) MasterMix containing 1 μg of Quantiscript RT, 4 μg of Quantiscript RT Buffer, and 1 μg of RT Primer Mix per sample was added to the sample after incubation. A cDNA synthesis reaction thermal cycling program was performed following the instructions of the manufacturer. cDNA samples were diluted with 180 μl of RNAse-free water to achieve a final concentration of 5 ng/μl. After mixing 10 ng of cDNA, 2.8 μl of RNAse-free water, 5 μl of MasterMix PerfeCTa SYBR Green (Applied Biosystems), and 0.2 μl of 10-μM forward and reverse primer mix of β-actin, a total volume of 10 μl was added to each well of a MicroAmp optical 384-well reaction plate (Applied Biosystems). Quantitative real-time polymerase chain reaction (qRT-PCR) reactions were then amplified in a QuantStudio 7 Flex System (Applied Biosystems).
Unless otherwise stated, all experiments were performed independently at least three times and the results for continuous variables are presented as means ± standard deviation. Statistical significance was determined using the Mann–Whitney test. Data were analyzed using Prism version 7 (GraphPad Software Inc., USA). P < 0.05 was considered significant for all tests.
| Results|| |
Effects of timing when processing fresh samples
Twenty patients were enrolled for sample viability and RNA quality tests. Ten RCC patients received nephrectomy and ten UTUC patients received nephroureterectomy. The main characteristics of the enrolled patients are detailed in [Table 1]. Viability tests and RNA extraction were performed 30, 45, and 60 min after the samples were resected. The mean percentages of live cells, which were assessed by flow cytometry [Figure 1]b, were 95.2%, 88.7%, and 67.8% for samples processed at 30, 45, and 60 min, respectively. We observed a significant decrease in cell viability when samples were processed more than 30 min after resection [30 vs. 45 min; P < 0.0001; [Figure 2]]. In RNA quality evaluations, there was no difference in the purity of RNA between groups. However, the RNA IQ number was correlated with the processing time points. RIN was better when the samples were processed earlier [30 vs. 45 min; P = 0.003; [Figure 2]]. Total RNA yield [30 vs. 45 min; P = 0.0081; [Figure 2]] and the amount of β-actin mRNA [30 vs. 45 min; P = 0.0015; [Figure 2]] also decreased when we processed the samples late. No change was noticed in RNA purity [30 vs. 45 min; P = 0.4213; [Figure 2]].
|Figure 2: Cell viability and RNA quality assessment in fresh tumors at different time points; (a) Comparison of percentage of cell viability between different time points; (b) Comparison of RNA IQ score between different time points; (c) Comparison of OD 260/280 ratio between different time points; (d) Comparison of total RNA yield between different time points; (e) Comparison of β-actin mRNA between different time points. N = 20 in each group; *P < 0.05; **P < 0.01; *** P < 0.001|
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Effects of timing when freezing samples
The same 20 samples were snap frozen 30, 45, and 60 min after they were resected. When RNA extraction was performed 1 month after sample collection, we discovered a significant difference between the RNA IQ counts of the different time points [30 vs. 45 min; P = 0.0019; [Figure 3]]. No significant difference was observed between groups in terms of RNA purity [30 vs. 45 min; P = 0.9650; [Figure 3]]. Total RNA yield [30 vs. 45 min; P = 0.0017; [Figure 2]] and the amount of β-actin mRNA [30 vs. 45 min; P = 0.0151; [Figure 3]] also decreased when we processed the samples later. Interestingly, the RNA quality detected from samples snap frozen at 30 min was not inferior to that detected from freshly extracted matched samples [IQ; 30 vs. 45 min; P = 0.6330; [Figure 4]]. RNA yield was also similar between the two groups [total RNA yield; 30 vs. 45 min; P = 0.2373; [Figure 4]].
|Figure 3: RNA quality assessment in snap-frozen tumors at different time points; (a) Comparison of RNA IQ score between different time points; (b) Comparison of OD 260/280 ratio between different time points; (c) Comparison of total RNA yield between different time points; (d) Comparison of β-actin mRNA between different time points. N = 20 in each group; *P < 0.05; **P < 0.01; *** P < 0.001|
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|Figure 4: RNA quality assessment in fresh and snap-frozen tumor samples; (a) Comparison of RNA IQ score; (b) Comparison of OD 260/280 ratio; (c) Comparison of total RNA yield; (d) Comparison of β-actin mRNA. N = 20 in each group; *P < 0.05; **P < 0.01; *** P < 0.001|
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Comparison of the effects of two different surgical techniques
Fifteen UC patients underwent transurethral resection of bladder tumors in our cohort. Transurethral tumor biopsy was performed before transurethral electroresection was performed. Both samples were collected and processed within 30 min. Viability tests showed a significant difference between the two groups [30 vs. 45 min; P < 0.0001; [Figure 5]]. Furthermore, in the RNA studies, no purity difference was noted between the groups [30 vs. 45 min; P = 0.3954; [Figure 5]]. Nevertheless, the RIN was higher in samples collected by transurethral biopsy [30 vs. 45 min; P < 0.0001; [Figure 5]]. When samples were resected using transurethral electroresection, we observed a decrease in total RNA yield [30 vs. 45 min; P = 0.0010; [Figure 5]] and the amount of β-actin mRNA [30 vs. 45 min; P = 0.0029; [Figure 5]].
|Figure 5: Cell viability and RNA quality assessment in fresh tumors resected by different techniques; (a) Comparison of percentage of cell viability; (b) Comparison of RNA IQ score; (c) Comparison of OD 260/280 ratio; (d) Comparison of total RNA yield; (e) Comparison of β-actin mRNA. Electro-, Transurethral electroresection; N = 15 in each group; *P < 0.05; **P < 0.01; *** P < 0.001|
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|Figure 6: Cell viability and RNA quality assessment in fresh tumors resected by different techniques; (a) Comparison of percentage of cell viability; (b) Comparison of the 260/280 nm absorbance ratioComparison of the RNA IQ score; (c) Comparison of the RNA IQ score Comparison of the OD 260/280 nm absorbance ratio; (d) Comparison of total RNA yield; (e) Comparison of β-actin mRNA. Electro-, Transurethral electro-resection; N=15 in each group; *P<0.05; **P<0.01; ***P<0.001|
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| Discussion|| |
The accessibility of viable human tissue samples has become crucial to supporting translational research focused on individualized tailored cancer therapy. However, fresh viable human tissues are rare and their use is time sensitive; moreover, the processing of these samples is complicated in real time. Normally, during surgery, tumor tissues are resected and placed beside the patient until the operation is complete and then the surgeon begins to evaluate the tumor and collects the ideal part for further research in the laboratory. This process potentially compromises the viability of the cells researchers use for further analyses. Hence, we conducted this study to determine the timing of fresh tumor sample collection. Our results showed clearly that fresh samples processed within 30 min have better viability, which indicates the importance of collecting tumor lesions as quickly as possible.
Furthermore, appropriate storage of these tissues with intact DNA and RNA for use in studies is an important objective for researchers. RNA is considered a highly fragile molecule; hence, avoiding RNA degradation is ultimate major challenge in this process. In the present study, we also aimed to identify the optimal timing at which to perform RNA extraction and preservation for high-quality RNA. We assessed RNA integrity by calculating the RIN. A traditional method for assessing RIN is agarose gel electrophoresis;, however, the Qubit RNA IQ Assay has also been shown to be a reliable method in previous publications.,, RNA quality IQ scores are presented by Qubit; larger scores indicate samples that contain mainly large RNA and vice versa. The utility of RNA obtained from tissue samples for further analyses can also be measured by PCR amplification of a specific sequence. Housekeeping genes expressed in all cells, such as β-actin, with little qPCR product suggest poor quality RNA and tissue degradation. Thus, we checked the quality of RNA with the following four measures: a Qubit™ RNA IQ assay, OD 260/280 ratio, total RNA yield, and the amount of β-actin mRNA. Three time points were checked: 30, 45, and 60 min. When processed within 30 min, most analyzed samples presented IQ values indicating a lack of adverse effects. Results also indicated greater RNA yields if RNA extraction was performed within 30 min of tumor tissue collection. Subsequent to 30 min, RNA was significantly degraded. Despite degradation of RNA, the OD 260/280 ratio was not affected, indicating that both materials are ideal in terms of purity.,, This suggested that protein contaminations are not associated with the timing of RNA extraction.
Currently, fresh frozen tissues (FFTs) are used widely in molecular profiling research and other related studies, especially retrospective analyses. Indeed, FFT can be used for transcriptomics, genomics, and proteomics. The use of FFT is also considered the best cryopreservation method, and it is widely accepted as a method for RNA preservation., In agreement with our findings, this approach was demonstrated to significantly increase RNA yield while maintaining RNA quality for further molecular applications. Furthermore, the freezing process avoids more complex procedures that researchers may immediately encounter when obtaining samples. If more widely applied, the accessibility of human tumor tissues to researchers will substantially improve and facilitate many basic, preclinical, and translational studies. Therefore, we evaluated the ideal timing for snap-freezing fresh samples, which could be used in future studies. Intriguingly, RNA appeared to be degraded without negative effects on protein contamination when snap freezing was completed within 30 min. The OD 260/280 values of most samples were around 1.8 and 2.0, which implies that the nucleic acids retrieved reached high purity levels. This confirms that a main reason for not realizing high-yield and good-quality RNA is the timing of sample freezing, even though the samples are not fresh. Thus, snap freezing should be completed quickly after the tumor samples are resected if RNA cannot be extracted immediately. This issue is particularly important in the current age of molecular oncology.
The cell viability and RNA quality of samples collected by different surgical techniques were also assessed in the present study. Intriguingly, transurethral electroresection, which is the standard procedure for bladder tumor removal, presented dissatisfying results compared to those obtained by transurethral tumor biopsy. This suggests that electrohyperthermia could damage the tumor and cells, which in turn would adversely affect the sample viability. Thus, transurethral electroresection is not an ideal procedure for the collection of samples intended for further molecular oncology studies.
| Conclusions|| |
Many variables must be considered when collecting fresh tumor samples including the tumor harvest method, sample processing method, and storage of the samples. Here, we provide evidence indicating that tumor tissue samples obtained and processed within 30 min have the viability and RNA quality necessary for their use in translational molecular research. Furthermore, to collect tissues from the urinary bladder, a transurethral biopsy of bladder tumor is considered a better method than a transurethral electroresection.
Financial support and sponsorship
This study was supported by grants from the Ministry of Science and Technology (MOST 107-2314-B-016-039-MY3, MOST 107-2314-B-016-024, and MOST 108-2314-B-016-012), from the Tri-Service General Hospital Research Foundation (TSGH-C107-008-S01, TSGH-C108-007-008-S01, TSGH-C01-109012, and TSGH-D-109068), from the Ministry of National Defence-Medical Affairs Bureau (MAB-106-107, MAB-107-082, MAB-107-076, MAB-108-076, and MAB-108-075), Taipei, Taiwan, R.O.C., and from the VGH, TSGH, AS Joint Research Program (VTA105-T-2-1, VTA106-T-4-1, and VTA107-T-2-1).
Conflicts of interest
Associate Prof. Ming-Hsin Yang, Associate Prof. En Meng, Dr. Tai-Lung Cha, Dr. Guang-Huan Sun and Dr. Dah-Shyong Yu, editorial board members at Urological Science, had no roles in the peer review process of or decision to publish this article. The other authors declared no conflicts of interest in writing this paper.
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