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Measurement of 2-methylthio Modifications in Mitochondrial Transfer RNAs by Reverse-transcription Quantitative PCR
反转录定量聚合酶链反应(qRT-PCR)法测量线粒体转运RNA中的2-甲硫基修饰   

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Abstract

2-Methylthio-N6-isopentenyladenosine (ms2i6A) is an evolutionally conserved posttranscriptional modification found at position 37 of four mammalian mitochondrial tRNAs, mt-tRNASer(UCN), mt-tRNATrp, mt-tRNAPhe and mt-tRNATyr. The ms2 modification in ms2i6A strengthens codon-anticodon interaction and contributes to accurate and efficient decoding. Deficiency of ms2 modifications impairs mitochondrial protein synthesis, which ultimately leads to the development of myopathy in mice and patients having mitochondrial diseases. Therefore, the level of ms2 could be utilized as an indicator that reflects the status of mitochondrial protein synthesis. Here, we describe a simple and fast quantitative PCR-based method to measure the ms2 level in total RNA sample.

Keywords: TRNA(tRNA), Modification(修改), Mitochondria(线粒体)

Materials and Reagents

  1. Total RNA (200 ng~1 ng)
    Note: We have been using 50~200 ng RNA as the starting materials. To compare the results between experiments, it is recommended to use same amount of RNA in all experiments.
    It is recommended to use a method that can purify small RNAs [e.g., TRIzol (Invitrogen, catalog number: 15596-018 ) or miRNeasy Mini Kit (QIAGEN, catalog number: 217004 ). Check the purity of RNA by a spectrophotometer. For consistency of experiments, always use RNA with an A260/A280 ratio > 1.8.
    Note: Currently, TRIzol is from “Thermo Fisher Scientific, AmbionTM, catalog number: 15596-018”.
  2. PCR grade distilled water
  3. Microtubes for PCR and real-time PCR (MicroAmp® Optical 8-Cap Strip) (Thermo Fisher Scientific, Applied Biosystems®, catalog number: 4323032 )
  4. DNase I (10x buffer is provided along with the enzyme) (Roche Diagnostics, catalog number: 04716728001 )
  5. Reverse transcription reagent (Transcriptor First Strand cDNA Synthesis kit) (Roche Diagnostics, catalog number: 04897030001 )
    Critical Point: It is recommended to use Transcriptor First Strand cDNA Synthesis kit for measurement of ms2 modification. Compared with other reverse transcription kits, this kit gives the highest dynamic range.
  6. Real-time PCR reagent (SYBR Premix Ex Taq II) (TAKARA BIO INC., catalog number: RR820S )
    Note: SYBR green based PCR reagents from other companies work equally well.
  7. Primers for detecting mitochondrial ms2 modifications in human RNA samples
    1. Hu_tRNAPhe primer f1: CTCCTCAAAGCAATACACTG
    2. Hu_tRNAPhe primer r1: AGCCCGTCTAAACATTTTCA
    3. Hu_tRNAPhe primer r2: GGGTGATGTGAGCCCGTCTA
    4. Hu_tRNASerUCN primer f1: GAGGCCATGGGGTTGG
    5. Hu_tRNASerUCN primer r1: CCCAAAGCTGGTTTCAAGC
    6. Hu_tRNASerUCN primer r2: AATCGAACCCCCCAAAGC
    7. Hu_tRNATrp primer f1: GGTTAAATACAGACCAAGAGC
    8. Hu_tRNATrp primer r1: CAACTTACTGAGGGCTTTGAA
    9. Hu_tRNATrp primer r2: TTAAGTATTGCAACTTACTGAGG
    10. Hu_tRNATyr primer f1: GCTGAGTGAAGCATTGGACT
    11. Hu_tRNATyr primer r1: AACCCCTGTCTTTAGATTTACA
    12. Hu_tRNATyr primer r2: AGAGGCCTAACCCCTGTCTT
  8. Primers for detecting mitochondrial ms2 modification in mouse RNA samples
    1. Ms_tRNAPhe primer f1: GCTTAATAACAAAGCAAAGCA
    2. Ms_tRNAPhe primer r1: TATCCATCTAAGCATTTTCA
    3. Ms_tRNAPhe primer r2: TGGGATACAATTATCCATCT
    4. Ms_tRNASerUCN primer f1: CATATAGGATATGAGATTGGC
    5. Ms_tRNASerUCN primer r1: AACCCCCTAAAATTGGTTTCA
    6. Ms_tRNASerUCN primer r2: GAAGGAATCGAACCCCCTAA
    7. Ms_tRNATrp primer f1: GGATATACTAGTCCGCGAGC
    8. Ms_tRNATrp primer r1: GTGTTTTCTTAGGGCTTTGA
    9. Ms_tRNATrp primer r2: GTTAAACTTGTGTGTTTTCTTAG
    10. Ms_tRNATyr primer f1: ATGGCTGAGTAAGCATTAGA
    11. Ms_tRNATyr primer r1: ACCTCTGTGTTTAGATTTAC
    12. Ms_tRNATyr primer r2: GAGGATTTAAACCTCTGTGT
      Notes:
      1. Standard PCR grade primers are sufficient for this protocol.
      2. The r1 primer is used for the measurement of total tRNA level, and the r2 primer is
        used for the measurement of ms2-modification level in individual tRNA.

Equipment

  1. Conventional PCR apparatus (Thermo Fisher Scientific, Applied Biosystems®, model: Veriti 96 Well Thermal Cycler )
  2. Real-time PCR apparatus (Life Technologies, Applied Biosystems®, model: 7300 Real Time PCR System )
    Note: Currently, it is “Thermo Fisher Scientific, model: 7300 Real Time PCR System ”.

Procedure

Part I. DNase treatment

  1. Prepare total RNA from cell and tissue samples of human or animals by TRIzol reagent or miRNeasy kit according to the manufacturer’s instructions.
  2. Mix following reagents in microtubes for DNase treatment on ice.
    Component
    Volume (µl/reaction)
    RNA (100 ng/µl)
    2
    DNase I
    0.5
    10x DNase I buffer
    2
    PCR grade water
    15.5
    Total volume
    20
    Note: For measurement of ms2 modification in multiple samples, it is recommended to prepare a master mixture of all reagents except RNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
  3. Put microtubes in PCR apparatus and use the following program.
    Time and temperature
    Digestion
    Inactivation
    Hold
    37 °C for 20 min
    75 °C for 10 min
    4 °C

  4. Use DNase I-treated sample for subsequent reverse transcription or store at -80 °C.

Part II. Reverse transcription


Figure 1. Workflow of the method to detect ms2 modification.
The mitochondrial tRNA is reversely transcribed by r1 primer and r2 primer, respectively. Because of the inhibitory effect of ms2-modification to the reverse transcription, the amount of cDNA generated by r2 primer (Green lines) highly depends on the ms2 levels in a given RNA sample. On the other hand, the cDNA generated by r1 primer (Blue lines) is independent of ms2 level, and could be utilized as an internal control. The amount of each cDNA is quantified by a subsequent quantitative PCR (qPCR) using f1 and r1 primers.


  1. For each tRNA species, mix DNase I-digested RNA with primer r1 or primer r2 on ice.
    Component
    Volume (µl/reaction)

    Sample (r1)
    Sample (r2)
    DNase-digested RNA
    2.5
    2.5
    Primer r1 (10 µM)
    1

    Primer r2 (10 µM)

    1
    Water
    3
    3
    Total volume
    6.5
    6.5
    Note: It is recommended to prepare a master mixture of all reagents except RNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
  2. Denature RNA at 65 °C for 10 min and immediately put samples on ice for 1 min.
  3. Add RT enzyme and buffer included in the Transcriptor First Strand cDNA Synthesis kit as follows on ice.
    Component
    Volume (µl/reaction)

    Sample (r1)
    Sample (r2)
    Denatured RNA
    6.5
    6.5
    5x buffer
    2
    2
    RNase inhibitor
    0.25
    0.25
    dNTP Mix
    1
    1
    Enzyme
    0.25
    0.25
    Total volume
    10
    10
    Note: It is recommended to prepare a master mixture of all reagents except RNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
  4. Perform reverse transcription according to the following program.
    Time and Temperature
    Reaction
    Inactivation
    Hold
    55 °C for 30 min
    85 °C for 5 min
    4 °C

  5. Samples are ready for quantitative PCR. Otherwise, samples can be stored at -20 °C.

Part III. Quantitative PCR

  1. Mix reagents as follows. In case other SYBR green-based PCR kits are used, follow the manufacturer' instructions as recommended.
    Component
    Volume (µl/reaction)

    Sample (r1)
    Sample (r2)
    SYBR Premix Ex Taq
    10
    10
    Primer f1 (10 µM)
    0.4
    0.4
    Primer r1 (10 µM)
    0.4
    0.4
    ROX reference dye
    0.4
    0.4
    cDNA
    2
    2
    Water
    6.8
    6.8
    Total volume
    20
    20
    Note: It is recommended to prepare a master mixture of all reagents except cDNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
  2. Set the program as follows and run real-time PCR. Add dissociation stage if required.
    Time and temperature
    First step
    PCR (40 cycles)
    Heat-activation
    Denaturing
    Annealing/Elongation
    Hold
    Cycle
    95 °C for 30 sec
    95 °C for 5 sec
    60 °C for 31 sec

Part IV. Data analysis
Analyze data from Samples (r1) and (r2) to obtain Ct values. The results obtained Samples (r1) and (r2) represents the total tRNA level and ms2-modification level in individual tRNA, respectively.
The normalized modification level in any given RNA sample is calculated as dCt = Ct (r2) – Ct (r1). Because the dCt value precisely reflects the modification level (Xie et al., 2013), the dCt value could be directly used for comparison of modification levels between multiple samples (see below for the representative data).

Representative data


Figure 2. Measurement of ms2 level of mt-tRNATyr in wild-type mouse heart tissue. Total RNA was purified from mouse heart tissue using TRIzol followed by 2-propanol precipitation. RNA sample was adjusted to 100 ng/µl and subjected to analysis as described above. The representative amplification curves using r1 primer (green) and r2 primer (blue) are shown. Ct (r2) = 14.8, Ct (r1) = 10.7. Modification level (dCt) in wild-type mouse heart = 14.8-10.7 = 4.1.


Figure 3. Measurement of ms2 level of mt-tRNATyr in heart tissue of Cdk5rap1 KO mouse. Total RNA was purified from heart tissue of Cdk5rap1 KO mouse that does not contain ms2 modification. RNA sample was adjusted to 100 ng/µl and subjected to analysis as described above. The representative amplification curves using r1 primer (red) and r2 primer (blue) are shown. Ct (r2) = 10.4, Ct (r1) = 10.4. Modification level (dCt) in Cdk5rap1 KO mouse heart = 10.4-10.4 = 0.


Figure 4. Comparison of the modification levels of mt-tRNATyr in heart tissues of wild-type (WT) and Cdk5rap1 KO mouse

Acknowledgments

The concept of this protocol was adapted from our previous study, in which the quantitative PCR was used to measure ms2 modification in cytosolic tRNALys(UUU) (Xie et al., 2013). We have successfully used this protocol to measure ms2 modification mitochondrial tRNA in our recent study (Wei et al., 2015). This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Wei, F. Y., Zhou, B., Suzuki, T., Miyata, K., Ujihara, Y., Horiguchi, H., Takahashi, N., Xie, P., Michiue, H., Fujimura, A., Kaitsuka, T., Matsui, H., Koga, Y., Mohri, S., Suzuki, T., Oike, Y. and Tomizawa, K. (2015). Cdk5rap1-mediated 2-methylthio modification of mitochondrial tRNAs governs protein translation and contributes to myopathy in mice and humans. Cell Metab 21(3): 428-442.
  2. Xie, P., Wei, F. Y., Hirata, S., Kaitsuka, T., Suzuki, T., Suzuki, T. and Tomizawa, K. (2013). Quantitative PCR measurement of tRNA 2-methylthio modification for assessing type 2 diabetes risk. Clin Chem 59(11): 1604-1612.

简介

2-甲硫基 - N 6 - 叔丁基腺苷(ms)是一种进化保守的转录后修饰发现 在四个哺乳动物线粒体tRNA,mt-tRNA Ser(UCN),mt-tRNA Trp ,mt-tRNA < tRNA Tyr 。 ms 2 6中的ms 2修饰加强了密码子 - 反密码子相互作用,并有助于准确和有效的解码。 缺乏修复损害线粒体蛋白质合成,其最终导致小鼠和患有线粒体疾病的患者中的肌病的发展。 因此,ms 2的水平可以用作反映线粒体蛋白质合成状态的指标。 在这里,我们描述了一种简单和快速的定量PCR为基础的方法来测量总RNA样本中的ms 2水平。

关键字:tRNA, 修改, 线粒体

材料和试剂

  1. 总RNA(200 ng?1 ng)
    注意:我们使用50?200ng RNA作为起始材料。为了比较实验之间的结果,建议在所有实验中使用相同量的RNA。
    推荐使用可纯化小RNA的方法。 TRIzol(Invitrogen,目录号:15596-018)或miRNeasy Mini Kit(QIAGEN,目录号:217004)。通过分光光度计检查RNA的纯度。为了实验的一致性,始终使用具有A 260 /A ratio> 1.8。
    注意:目前, -018"。
  2. PCR级蒸馏水
  3. 用于PCR和实时PCR的Microtubes(MicroAmp光学8-Cap条)(Thermo Fisher Scientific,Applied Biosystems ,目录号:4323032)
  4. DNase I(与酶一起提供10x缓冲液)(Roche Diagnostics,目录号:04716728001)
  5. 逆转录试剂(Transcriptor First Strand cDNA Synthesis kit)(Roche Diagnostics,目录号:04897030001)
    关键点:建议使用Transcriptor第一链cDNA合成试剂盒测量ms 2 修饰。与其他逆转录试剂盒相比,该试剂盒具有最高的动态范围。
  6. 实时PCR试剂(SYBR Premix Ex Taq II)(TAKARA BIO INC。,目录号:RR820S)
    注意:来自其他公司的SYBR green基PCR试剂同样良好。
  7. 用于检测人RNA样品中线粒体ms 2修饰的引物
    1. Hu_tRNA Phe 引物f1:CTCCTCAAAGCAATACACTG
    2. Hu_tRNA Phe 引物r1:AGCCCGTCTAAACATTTTCA
    3. Hu_tRNA Phe 引物r2:GGGTGATGTGAGCCCGTCTA
    4. Hu_tRNA SerUCN 引物f1:GAGGCCATGGGGTTGG
    5. Hu_tRNA SerUCN 引物r1:CCCAAAGCTGGTTTCAAGC
    6. Hu_tRNA SerUCN 引物r2:AATCGAACCCCCCAAAGC
    7. Hu_tRNA Trp 引物f1:GGTTAAATACAGACCAAGAGC
    8. Hu_tRNA Trp 引物r1:CAACTTACTGAGGGCTTTGAA
    9. Hu_tRNA Trp 引物r2:TTAAGTATTGCAACTTACTGAGG
    10. Hu_tRNA Tyr 引物f1:GCTGAGTGAAGCATTGGACT
    11. Hu_tRNA Tyr 引物r1:AACCCCTGTCTTTAGATTTACA
    12. Hu_tRNA Tyr 引物r2:AGAGGCCTAACCCCTGTCTT
  8. 用于检测小鼠RNA样品中线粒体ms 2 修饰的引物
    1. Ms_tRNAPhe引物f1:GCTTAATAACAAAGCAAAGCA
    2. Ms_tRNAPhe引物r1:TATCCATCTAAGCATTTTCA
    3. Ms_tRNAPhe引物r2:TGGGATACAATTATCCATCT
    4. Ms_tRNA SerUCN 引物f1:CATATAGGATATGAGATTGGC
    5. Ms_tRNA SerUCN 引物r1:AACCCCCTAAAATTGGTTTCA
    6. Ms_tRNA SerUCN 引物r2:GAAGGAATCGAACCCCCTAA
    7. Ms_tRNA Trp 引物f1:GGATATACTAGTCCGCGAGC
    8. Ms_tRNA Trp 引物r1:GTGTTTTCTTAGGGCTTTGA
    9. Ms_tRNA Trp 引物r2:GTTAAACTTGTGTGTTTTCTTAG
    10. Ms_tRNA Tyr 引物f1:ATGGCTGAGTAAGCATTAGA
    11. Ms_tRNA Tyr 引物r1:ACCTCTGTGTTTAGATTTAC
    12. Ms_tRNA Tyr 引物r2:GAGGATTTAAACCTCTGTGT
      注意:
      1. 标准PCR等级引物对于该方案是足够的。
      2. r1引物用于测量总tRNA水平,r2引物是。
        用于测量个体tRNA中的ms 2 - 修饰水平。

设备

  1. 常规PCR装置(Thermo Fisher Scientific,Applied Biosystems ,型号:Veriti 96 Well Thermal Cycler)
  2. 实时PCR装置(Life Technologies,Applied Biosystems ,型号:7300实时PCR系统)
    注意:目前,它是"Thermo Fisher Scientific,型号:7300实时PCR系统"。

程序

DNase处理

  1. 从人或动物的细胞和组织样品制备总RNA TRIzol试剂或miRNeasy试剂盒 指令。
  2. 将以下试剂混合在微管中,在冰上进行DNase处理
    组件
    体积(μl/反应)
    RNA(100ng /μl)
    2
    DNase I
    0.5
    10x DNA酶I缓冲液
    2
    PCR级水
    15.5
    总量
    20
    注意:为了测量多个样本中的ms 2 修改, 建议制备除RNA以外的所有试剂的主混合物 减少变异性。当制备主混合物时,通常是 希望准备稍微过量以补偿移液 不准确。
  3. 将微管放入PCR仪中,使用以下程序
    时间和温度
    消化
    停用
    暂停
    37℃20分钟
    75℃10分钟
    4°C

  4. 使用DNase I处理的样品进行随后的逆转录或储存在-80°C。

第二部分。反转录


图1.检测ms 2 修饰的方法的工作流程。线粒体tRNA分别由r1引物和r2引物反转录。由于rs2引物对逆转录的抑制作用,由r2引物(绿线)产生的cDNA的量高度依赖于rs2引物中的ms 2水平。给定RNA样品。另一方面,由r1引物(蓝线)产生的cDNA独立于ms 2水平,并且可以用作内部对照。通过使用f1和r1引物的随后的定量PCR(qPCR)定量每种cDNA的量。


  1. 对于每种tRNA种类,将DNA酶I消化的RNA与引物r1或引物r2在冰上混合
    组件
    体积(μl/反应)

    示例(r1)
    示例(r2)
    DNase消化的RNA 2.5
    2.5
    引物r1(10μM)
    1
    -
    引物r2(10μM)
    -
    1

    3
    3
    总量
    6.5
    6.5
    注意:建议准备所有试剂的主混合物 除了RNA以减少变异性。当制备主混合物时,它是 ?通常需要制备稍微过量以补偿 移液不准确。
  2. 使RNA在65℃变性10分钟,并立即将样品在冰上放置1分钟
  3. 如下在冰上加入包含在Transcriptor第一链cDNA合成试剂盒中的RT酶和缓冲液 < table height ="167"border ="1"bordercolor ="#000000"cellpadding ="2"cellspacing ="0" width ="310"> 组件
    体积(μl/反应)

    示例(r1)
    示例(r2)
    变性RNA
    6.5
    6.5
    5x缓冲区
    2
    2
    核糖核酸酶抑制剂
    0.25
    0.25
    dNTP Mix
    1
    1

    0.25
    0.25
    总量
    10
    10
    注意:建议准备所有试剂的主混合物 除了RNA以减少变异性。当制备主混合物时,它是 ?通常需要制备稍微过量以补偿 移液不准确。
  4. 根据以下程序执行逆转录。
    时间和温度
    反应
    停用
    暂停
    55℃30分钟
    85℃5分钟
    4°C

  5. 样品准备用于定量PCR。否则,样品可以储存在-20°C。

第三部分。定量PCR

  1. 混合试剂如下。如果使用其他SYBR基于绿色的PCR试剂盒,请按照制造商的说明推荐
    组件
    体积(μl/反应)

    示例(r1)
    示例(r2)
    SYBR Premix Ex Taq
    10
    10
    引物f1(10μM)
    0.4
    0.4
    引物r1(10μM)
    0.4
    0.4
    ROX参照染料
    0.4
    0.4
    cDNA
    2
    2

    6.8
    6.8
    总量
    20
    20
    注意:建议准备所有试剂的主混合物 除了cDNA以减少变异性。当制备主混合物时 通常期望制备稍微过量以补偿 移液不准确。
  2. 设置程序如下,并运行实时PCR。如果需要,添加解离阶段。
    时间和温度
    第一步
    PCR(40个循环)
    热激活
    变性
    退火/延伸
    暂停
    循环
    95°C 30秒
    95°C 5秒
    60℃,31秒

第四部分。数据分析
分析样品(r1)和(r2)的数据以获得Ct值。所获得的结果样品(r1)和(r2)分别代表个体tRNA中的总tRNA水平和ms 2修饰水平。
任何给定RNA样品中的归一化修饰水平计算为dCt = Ct(r2)-Ct(r1)。因为dCt值精确地反映修饰水平(Xie等人,2013),所以dCt值可以直接用于多个样品之间的修饰水平的比较(关于代表性数据,参见下文)。

代表数据


图2.测量ms 2 mt-tRNA水平 Tyr 在野生型小鼠心脏组织中。 使用TRIzol从小鼠心脏组织纯化总RNA,然后2-丙醇沉淀。 RNA样品调节至100ng /μl,并如上所述进行分析。显示了使用r1引物(绿色)和r2引物(蓝色)的代表性扩增曲线。 Ct(r2)= 14.8,Ct(r1)= 10.7。野生型小鼠心脏中的修饰水平(dCt)= 14.8-10.7 = 4.1。


图3.测量ms 2 mt-tRNA水平 Tyr 在 Cdk5rap1 KO m 的心脏组织中<不含有ms 2 修饰的Cdk5rap1 KO小鼠。 RNA样品调节至100ng /μl,并如上所述进行分析。显示了使用r1引物(红色)和r2引物(蓝色)的代表性扩增曲线。 Ct(r2)= 10.4,Ct(r1)= 10.4。 Cdk5rap1 KO小鼠心脏中的修饰水平(dCt)= 10.4-10.4 = 0.

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图4.野生型(WT)和Cd k5rap1 KO小鼠的心脏组织中mt-tRNA Tyr 的修饰水平的比较。

致谢

该方案的概念改编自我们以前的研究,其中定量PCR用于测量细胞溶质tRNA(UUU)中的ms 2修饰 et al。,2013)。在我们最近的研究中,我们已经成功地使用该方案来测量ms 2 修饰线粒体tRNA(Wei等人,2015)。这项工作得到了日本教育,文化,体育,科学和技术部的科学研究资助。

参考文献

  1. Wei,FY,Zhou,B.,Suzuki,T.,Miyata,K.,Ujihara,Y.,Horiguchi,H.,Takahashi,N.,Xie,P.,Michiue,H.,Fujimura,A.,Kaitsuka ,T.,Matsui,H.,Koga,Y.,Mohri,S.,Suzuki,T.,Oike,Y.and Tomizawa,K。(2015)。 Cdk5rap1介导的线粒体tRNA的2-甲硫基修饰控制蛋白质翻译并促进小鼠和人类的肌病。 Cell Metab 21(3):428-442。
  2. Xie,P.,Wei,F.Y.,Hirata,S.,Kaitsuka,T.,Suzuki,T.,Suzuki,T.and Tomizawa,K。(2013)。 用于评估2型糖尿病风险的tRNA 2-甲硫基修饰的定量PCR测量。 em> Clin Chem 59(11):1604-1612。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wei, F. and Tomizawa, K. (2016). Measurement of 2-methylthio Modifications in Mitochondrial Transfer RNAs by Reverse-transcription Quantitative PCR. Bio-protocol 6(1): e1695. DOI: 10.21769/BioProtoc.1695.
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