|Year : 2018 | Volume
| Issue : 1 | Page : 77-80
Comparison of different methods of RNA preparation from peripheral blood for nucleic acid amplification assay
Dong Eun Lee1, Hanna Lee2, Seon Duk Lee2, Hyung Soo Han2, Jae Young Choe1, Sora Yun3, Hong-Jun Park3, Jungbae Park1, Jong Kun Kim1
1 Department of Emergency Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea
2 Department of Physiology, School of Medicine, Kyungpook National University, Daegu, South Korea
3 Mmonitor Inc., Daegu, South Korea
|Date of Web Publication||2-May-2018|
Prof. Jong Kun Kim
Department of Emergency Medicine, School of Medicine, Kyungpook National University, 680 Gukchaebosang-ro, Jung-gu, Daegu 41944
Source of Support: None, Conflict of Interest: None
Background: Nucleic acid amplification assays (NAAs), such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are used for disease diagnosis. Current nucleic acid isolation kits require several hours for completion of protocol including the complicated handling steps. Objective: In this study, a simple and cost-effective nucleic acid preparation method was developed, and its performance was compared with those of commercial kits. Materials and Methods: RNA was prepared using our method and three commercial RNA isolation kits. The RNA quantity and quality were evaluated using the NanoDrop spectrophotometer and Agilent 2100 bioanalyser. Reverse transcription LAMP (RT-LAMP) reactions were performed to determine the usability of the RNA preparation methods. Results: The concentrations of RNA extracted from blood samples by four different methods were sufficient for use in NAAs. The RNA integrity number was >7.0 when RNA was isolated using other RNA isolation kits but lower when prepared using our method. The RT-LAMP reaction was successfully performed when RNA was prepared using any of the methods. Conclusions: These results demonstrate that despite the lower purity and integrity of RNA, our RNA preparation protocol is simple and rapid and shows reasonable performance in RT-LAMP.
Keywords: Loop-mediated isothermal amplification, nucleic acid amplification assays, RNA preparation, whole blood
|How to cite this article:|
Lee DE, Lee H, Lee SD, Han HS, Choe JY, Yun S, Park HJ, Park J, Kim JK. Comparison of different methods of RNA preparation from peripheral blood for nucleic acid amplification assay. Indian J Med Microbiol 2018;36:77-80
|How to cite this URL:|
Lee DE, Lee H, Lee SD, Han HS, Choe JY, Yun S, Park HJ, Park J, Kim JK. Comparison of different methods of RNA preparation from peripheral blood for nucleic acid amplification assay. Indian J Med Microbiol [serial online] 2018 [cited 2019 Feb 23];36:77-80. Available from: http://www.ijmm.org/text.asp?2018/36/1/77/231679
| ~ Introduction|| |
Nucleic acid amplification assays (NAAs), such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), are used for disease diagnosis.,,, Most of the NAAs use pure nucleic acids isolated from samples as amplification templates as they enhance the performance of NAAs. Current nucleic acid isolation kits require several hours for completion of protocol, including the complicated handling steps, which can result in incorporation of human errors depending on the dexterity of the sample handler. Recent years have seen the advent of high-performance and high-throughput automated nucleic acid preparation machines which, however, are not suitable for small-scale laboratories or for point of care use such as in outpatient clinics. In fact, nucleic acid extraction procedures can impede the use of NAAs in urgent clinical settings despite their high sensitivity and specificity compared to rapid immunoassays. Therefore, convenient nucleic acid preparation methods are required to promote the use of NAAs in medicine and healthcare. In this study, a simple and cost-effective nucleic acid preparation method from peripheral blood samples was developed, and its performance was compared with those of commercial nucleic acid isolation kits.
| ~ Materials and Methods|| |
To prepare total RNA from whole blood samples, three commercial kits namely, PAXgene Blood RNA tubes (#762165) and PAXgene Blood RNA kit (#762174) from Qiagen (Seoul, South Korea), Tempus™ Blood RNA collection tubes (#4342792) and Tempus™ Spin RNA isolation kit (#4380204) from Thermo Fisher Scientific (Seoul, South Korea) and QIAamp RNA blood mini kit (#52304) from Qiagen (Seoul, South Korea) were used. The Miso ® RNA amplification kit from Monitor (Daegu, South Korea) was used for gene expression assay. Gene expression reactions were performed using human beta-actin and tumour necrosis factor (TNF)-alpha-specific primer sets shown in [Table 1]. All reagents were purchased from Sigma-Aldrich unless otherwise specified.
|Table 1: Primer sets used for reverse transcription loop.mediated isothermal amplification reactions|
Click here to view
Sample collection and RNA preparation
Fresh whole blood samples were collected from our hospital in accordance with the approved guidelines and relevant regulations. All participants provided their written informed consent before participating in the study. The Ethics Committee and Institutional Review Board of our hospital approved all experimental procedures. All experiments were performed in accordance with the approved guidelines.
A volume of 2 mL peripheral blood from 10 healthy people were collected in PAXgene Blood RNA tubes, and the RNA isolation was performed following the manufacturer's protocol provided with the PAXgene Blood RNA kit. Peripheral blood (2 mL) from 10 healthy people was collected in Tempus™ Blood RNA collection tubes, and the RNA isolation was performed following the manufacturer's manual provided with the Tempus™ Spin RNA isolation kit. Peripheral blood (2 mL) from 10 healthy people was collected in ethylenediaminetetraacetic acid (EDTA)-treated bottles and the RNA isolation was performed following the manufacturer's manual provided with the QIAamp RNA blood mini kit. For our RNA preparation method, 2 mL peripheral blood from 10 healthy people was collected in EDTA-treated bottles. A volume of 300 μL of EDTA-treated blood was transferred to a tube containing 3 mL buffer solution and mixed well followed by transfer of 300 μL mixed blood to another tube and incubation at 80°C for 10 min using a thermal Cycler from Bio-Rad (Seoul, South Korea). The boiled blood sample was centrifuged at 6000 × g for 10 min. After centrifugation, 2 μL supernatant was added to the reverse transcription LAMP (RT-LAMP) master mix per reaction.
Measurement of RNA quality
The quality of isolated RNA was measured by a NanoDrop spectrophotometer (Seoul, South Korea) and Agilent 2100 bioanalyser (Seoul, South Korea). The purity of the RNA samples was analysed using the NanoDrop spectrophotometer, which measured absorbance at 230, 260 and 280 nm. The 260/280 and 260/230 absorbance ratios were calculated to evaluate RNA purity. The Agilent 2100 bioanalyser was used to measure RNA integrity, which is displayed as the RNA integrity number (RIN) and the ratio of 28S and 18S rRNAs.
Gene expression assay
Gene expression was evaluated with one-step RT-LAMP using RNA samples prepared by four different methods. RT-LAMP was performed at 58°C for 30 min. Each assay was repeated thrice for all experimental conditions.
| ~ Results|| |
Optimisation of RNA preparation method
To determine the optimal experimental conditions, various parameters, such as incubation time and temperature, centrifugation speed and time, buffer components and blood dilution factor, were modified and evaluated.
Heating temperature was tested at 20°C, 60°C and 80°C, and the duration of heating was evaluated at 5 and 10 min [Figure S1]. RNA integrity decreased with increase in temperature since heating destroyed the nucleic acid structure. However, the cell membrane remained intact at lower temperatures and did not release nucleic acids into the preparation solution resulting in RNA concentration that was insufficient for use in NAA.
The effects of centrifugation speed (2000 and 6000 × g) and centrifugation duration (3, 5 and 10 min) on RNA yield were evaluated [Figure S2]. Higher speed and longer duration showed better separation of blood cells and heavy particles from the solution layer. Highly pure solutions showed less interference in NAA as haemoglobin and other blood components are known to inhibit DNA polymerase activity.
To constitute the basic preparation buffer, 8 g NaCl, 0.2 g KCl, 1.44 g Na2 HPO4 and 0.24 g KH2 PO4 were dissolved in distilled water and the pH was adjusted to 7.4 to attain a final volume of 1 litre. Different amounts of detergents such as Triton X-100 or sodium dodecyl sulphate were added in the basic preparation buffer solution to determine the performance of the preparation method. Contrary to our expectations, addition of detergents to the basic preparation buffer inhibited NAA reaction as shown in [Figure S3]a and [Figure S3]b. We expected the detergents to destroy the cell membrane of both red and white blood cells and release nucleic acids in the solution. While detergents assist in releasing nucleic acids from the nuclei, they also release haemoglobin and other inhibitors from blood cells, which reduce the efficiency of NAA.
The optimal dilution factor was determined by adding 500, 1,000, 2,000 and 3,000 μL preparation buffer to 300 μL of whole blood. In our assay, the dilution factor did not affect NAA performance [Figure S4]. The final preparation conditions were selected on the basis of the results of the optimisation experiments, which are indicated in the materials and methods, and the representative images of samples at each step are shown in [Figure S5].
Concentration and purity of RNA
RNA solution was prepared from whole blood using the PAXgene Blood RNA kit, Tempus™ Spin RNA isolation kit, QIAamp RNA blood mini kit and our method. The concentration and purity of the RNA solutions are shown in [Table 2]. The concentrations of the RNA solution prepared by the four methods were 99.2, 69.7, 23.6 and 50.1 (ng/μL), respectively. The 260/280 ratios of RNA isolated using these methods were approximately 2.0. The 260/230 ratio was higher for RNA isolated using the PAXgene Blood RNA kit and Tempus™ Spin RNA isolation kit than that of RNA prepared using the QIAamp RNA blood mini kit and our method.
Integrity of RNA
RNA solution was prepared from whole blood using the PAXgene Blood RNA kit, Tempus™ Spin RNA isolation kit, QIAamp RNA blood mini kit and our method. The integrity of the RNA solution is demonstrated in [Table 3]. The intensity ratios of the 28S and 18S rRNA bands were 1.5, 1.7, 1.2 and not available (N/A), whereas RIN were 9.0, 9.3, 7.7 and N/A, respectively.
Reverse transcription loop-mediated isothermal amplification reaction to detect human gene expression
Colour change was observed in all samples when RT-LAMP reaction was performed with human beta-actin [Figure 1] and TNF-alpha [Figure 2] as gene expression targets.
|Figure 1: Reverse transcription loop-mediated isothermal amplification reaction of different RNA isolation kits using human beta-actin. Colour change was observed in R1, R2, R3 and S1 samples. R1: PAXgene Blood RNA kit, R2: Tempus™ Spin RNA isolation kit, R3: QIAamp RNA Blood mini kit, S1: Our final method, S2: Our preliminary method, NC: Negative control, PC: Positive control|
Click here to view
|Figure 2: Reverse transcription loop-mediated isothermal amplification reaction of different RNA isolation kits using human tumour necrosis factor-alpha. Colour change was observed in R1, R2, R3 and S1 samples. R1: PAXgene Blood RNA kit, R2: Tempus™ Spin RNA isolation kit, R3: QIAamp RNA Blood mini kit, S1: Our final method, S2: Our preliminary method, NC: Negative control, PC: Positive control|
Click here to view
| ~ Discussion|| |
NAAs, including PCR or LAMP, have been recently used for diagnosing many diseases. Previous studies have used single chip-based methods to simplify the extraction, amplification and detection steps of nucleic acid isolation, and efforts have been made to apply them to the LAMP reaction.,, In the previous study, Choe et al. suggested that RT-LAMP has advantages of simplicity, cost-effectiveness and higher amplification efficiency when used for studying human gene expression. However, the nucleic acid extraction step of current protocols is too complicated and time-consuming to be used in busy clinical settings, and therefore, convenient methods of sample preparation are required.We developed a new method to circumvent this problem, and our nucleic acid preparation method was of reasonable quality compared to RNA extracted using other methods [Table 4].
The RNA concentration obtained with our method was sufficient to be used for the RT-LAMP assay. Even though the 'acceptable' level for nucleic acid purity might vary with downstream applications such as next-generation sequencing, PCR, LAMP and microarray, nucleic acids with 260/280 absorbance ratios between 1.8 and 2.0 are generally considered to be free of significant contamination. The 260/280 ratio of RNA isolated using our method was 1.99 whereas the 260/230 ratio was lower than those of RNA purified using other methods as our protocol lacks the purification step.
The RIN is a software tool that estimates the integrity of total RNA samples. To determine the RIN, the instrument software uses an algorithm that takes into account the entire electrophoretic trace of the RNA instead of the ratio of 28S and 18S rRNAs. The RIN scale ranges from 0 to 10, with 10 indicating maximum RNA integrity and a 28S: 18S rRNA ratio of 2:1 is generally representative of high-quality RNA. The RIN and the ratio of 28S and 18S rRNAs (determined by the Agilent 2100 bioanalyser) were N/A for our method. We expect that boiling at 95°C might have damaged RNA structure and integrity, leading to a poor outcome.
Nonetheless, the time required for the experimental steps in our method are shorter than those of other RNA preparation methods [Table 4].
| ~ Conclusions|| |
The basic concept of our method is to simplify the manipulation steps and reduce the time for whole blood sample preparation, while partially compromising on the quality of the final RNA sample for NAAs. The results of this study demonstrate that our RNA preparation method is simple, rapid and economical and yields nucleic acids of reasonable quality for amplification assays.
This work was financially supported by Biomedical Research Institute grant, Kyungpook National University Hospital (2015).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, et al.
Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350-4.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al.
Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:E63.
Johnson G, Ayers M, McClure SC, Richardson SE, Tellier R. Detection and identification of Bartonella
species pathogenic for humans by PCR amplification targeting the Riboflavin Synthase Gene (ribC). J Clin Microbiol 2003;41:1069-72.
Parida M, Sannarangaiah S, Dash PK, Rao PV, Morita K. Loop mediated isothermal amplification (LAMP): A new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol 2008;18:407-21.
Kopp MU, Mello AJ, Manz A. Chemical amplification: Continuous-flow PCR on a chip. Science 1998;280:1046-8.
Hataoka Y, Zhang L, Mori Y, Tomita N, Notomi T, Baba Y, et al.
Analysis of specific gene by integration of isothermal amplification and electrophoresis on poly (methyl methacrylate) microchips. Anal Chem 2004;76:3689-93.
Notomi T, Mori Y, Tomita N, Kanda H. Loop-mediated isothermal amplification (LAMP): Principle, features, and future prospects. J Microbiol 2015;53:1-5.
Choe JY, Han HS, Lee SD, Lee H, Lee DE, Ahn JY, et al.
A comparative study of three different gene expression analysis methods. Technol Health Care 2017;25:1073-80.
Manchester KL. Value of A260/A280 ratios for measurement of purity of nucleic acids. Biotechniques 1995;19:208-10.
Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, et al.
The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 2006;7:3.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]