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Extraction of Small RNA and qPCR Validation of miRNAs in Vigna mungo
黑吉豆中小RNA的提取和miRNA的定量PCR   

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Abstract

Small RNAs like microRNAs (miRNAs), small interfering RNAs (siRNAs) and other noncoding RNAs including snRNA and snoRNA have tremendous impact on eukaryotic gene regulation. Extraction of high quality small RNAs is an important prerequisite for experimental analyses of miRNAs. This will prevent RNA degradation and remove associated contaminations including polyphenols, polysaccharides and other secondary metabolites. In this protocol we describe a simple way to isolate small RNAs from the leaf tissues of Vigna mungo combining the protocols of two commercially available kits with some modifications.

Keywords: MicroRNA(microRNA), MYMIV(mymiv), Next Gen Sequencing(下一代基因测序), Vigna mungo(豇豆蒙哥)

Materials and Reagents

  1. Vigna mungo MYMIV-resistant recombinant inbred line, VMR84
  2. mirPremier microRNA isolation kit (Sigma-Aldrich, catalog number: SNC10 )
  3. Mir-X miRNA FirstStrand synthesis and SYBR qRT-PCR kit (Takara Bio Company, Clontech, catalog number: 638314 )
  4. 2-mercaptoethanol (Sisco Research Laboratories, catalog number: 1324196 )
  5. Ethanol (Merck, catalog number: K41540783 )
  6. Liquid nitrogen and dry ice
  7. RNase free water (Bangalore Genei, catalog number: 612151181001730 )
  8. Agarose (Sisco Research Laboratories, catalog number: 0140229 )
  9. Ethidium bromide (Sisco Research Laboratories, catalog number: 054817 )
  10. 1x TAE buffer (see Recipes)
  11. Ethidium bromide stock (see Recipes)

Equipment

  1. Thermocycler (DNA Engine Cycler, model: PTC-200 )
  2. Real-time qPCR (Biorad iQ5 Real-Time PCR Detection System)
  3. Centrifuge (Thermo Fisher Scientific, model: MicroCL21 )
  4. Electrophoresis apparatus (Bangalore Genei)
  5. Heat block or water bath
  6. Mortar and pestle
  7. NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)

Procedure

  1. Plant material and growth conditions
    Vigna mungo recombinant inbred line, VMR84, was used for isolation of small RNA.
    1. Mature seeds were surface sterilized with 0.1% HgCl2 for 10 min and rinsed twice in deionized water.
    2. Surface sterilized seeds were germinated in moistened filter papers at 28 ± 1 °C and 70% relative humidity and 16 h light and 8 h dark.
    3. Germinated seeds (after incubation of three days) were then transferred into sterile soil mix and grown in a greenhouse at 25 ± 1 °C. After 21 days of seedling growth (at 16 h light and 8 h dark), young trifoliate leaves were collected, frozen in liquid nitrogen and stored at -80 °C until RNA isolation.

  2. Small RNA isolation
    1. Small RNA was isolated from Vigna mungo tissues using mirPremier microRNA isolation kit according to the manufacturer’s instructions with some modifications.
    2. The harvested leaf tissue was grounded to a fine powder in liquid nitrogen using a mortar and pestle.
    3. 750 μl of the lysis buffer was added to the frozen tissue powder (80 mg), vortexed for 2 min and incubated at 55 °C for 5 min.
    4. The sample was then centrifuged at maximum speed for 5 min to remove cellular debris, genomic DNA, and large RNA.
    5. The lysate supernatant (800 μl approx.) was filtered through the filtration column and the flow through was mixed with 100% ethanol and mixed immediately.
    6. The lysate was then passed through binding column and centrifuged at a maximum speed for 30 sec and the flow-through liquid was decanted.
    7. After binding, the column was first washed with 700 μl of 100% ethanol and centrifuged at 14,000 x g for 30 sec and again the flow-through was discarded.
    8. The second wash was done by adding 500 μl of binding solution into the column and centrifuged at maximum speed (14,000 x g) for 1 min.
    9. Subsequently 500 ml of the ethanol-diluted wash solution 2 was added to the column for a third wash. After centrifugation at maximum speed (14,000 x g) for 30 sec, the flow-through was discarded.
    10. A second wash with ethanol-diluted wash solution 2 (500 ml) was performed and the flow-through liquid was discarded.
    11. Next the column was dried by centrifuging at maximum speed (14,000 x g) for 1 min. The column-tube assembly was carefully removed from the centrifuge to avoid splashing of the residual flow-through liquid to the dried column.
    12. Small RNA was eluted from the column using 50 ml elution solution and by centrifugation at 16,000 x g and the process was repeated to improve small RNA yield. The purified RNA was stored at -20 °C.
    13. Quantitative and qualitative analyses of RNA were done by NanoDrop with 1 μl of the sample followed by agarose gel electrophoretic separation. The ratio of absorbance at 260 to 280 nm, calculated by (A260 - A320)/(A280-A320), is typically between 1.8 and 2.2.

  3. First-strand cDNA synthesis
    Small RNAs were polyadenylated and reverse transcribed using the Mir-X miRNA First-Strand Synthesis kit following manufacturer’s instructions.
    1. Briefly, 5 μl mRQ buffer (2x), 5 μg RNA and 1.25 μl mRQ enzyme was mixed in a reaction volume of 10 μl and incubated in a thermocycler for 1 h at 37 °C, then terminate at 85 °C for 5 min to inactivate the enzymes.
    2. After reverse transcription, the cDNA was diluted by adding 90 μl nuclease free water to bring the total volume to 100 μl. The reverse transcribed cDNA is now ready for the miRNA quantification.

  4. Quantification of miRNA by qPCR
    Quantitative real time PCR was done using Mir-X miRNA qPCR SYBR Kit. The methodology is as follows:
    1. Designing of primers for qPCR: It is recommended to use the entire sequence of mature miRNA of desired or related plant species (available in mirBase) as miRNA specific, 5' primer. The 3' primer for qPCR is the mRQ 3' primer supplied with the kit.
    2. Amplification of miRNA by qPCR.
      1. The delta-delta Ct method was used to quantify the presence of each miRNA relative to the level of U6 snRNA, an internal control. For this experiment, qPCR amplification of U6 snRNA was done for each cDNA samples in duplicates. Additionally, inclusion of appropriate no template controls (NTC) for each primer set was performed to confirm absence of genomic DNA contamination.
      2. For each qPCR reaction, 20 μl PCR reaction mixture was prepared comprising of 1x SYBR advantage premix, 1x ROX dye, 0.2 mM of both forward and reverse primers and 50 ng of the first‐strand cDNA. For U6 reaction, the forward and reverse primers of U6, provided with the kit, were used.
      3. qPCR reactions were incubated in a 96 well plate at 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 sec and 60 °C for 20 sec. Amplification cycles were followed by a melting curve analysis ranging from 56 to 95 °C, with 0.5 °C temperature increasing at every 10 sec. Melting curve for each amplicon was observed carefully to confirm the specificity of the primers used. The threshold cycle (Ct) values were recorded.
    3. Data analysis and calculating miRNA levels using the Delta-Delta Ct (or ddCt) method.
      The ddCt method provides a measure of the relative levels of a miRNA between two samples by comparing them to a normalization standard. Here the miRNA and the U6 RNA are amplified for each sample to determine the Ct value. This allows relative levels to be determined using the ddCt calculation.
      The relative copy number is calculated using the ddCt method:
      [miR-X] Leaf/[miR-X]Stem: 2–dCt(Leaf)/ 2–dCt(Stem)
      However, precision of this method of quantification depends on the amplification efficiency of the primer pairs that should near 100% otherwise quantification will not be accurate.


      Figure 1. Melt curve analysis of mir156 produced during qPCR analysis. The graph shows a plot of negative derivative of fluorescence versus temperature (°C) for miRNA amplification. Presence of a single peak denotes nonspecific amplification of a single product.

Recipes

  1. 1x TAE (1 L)
    242 g Tris base (MW 121.1), 57.1 ml Glacial acetic acid 100 ml 0.5 M EDTA was added and mixed in 600 ml of ddH2O and the final volume was adjusted to 1 L.
    Next 20 ml of 50x TAE was added to 980 ml of ddH2O to prepare 1 L of 1x TAE (pH 8.3 at 25 °C).
  2. Ethidium bromide stock
    1 g of ethidium bromide was dissolved in 100 ml of ddH2O to prepare a stock of 10 mg/ml.

Acknowledgments

The original version of this protocol was described in Paul et al. (2014). This work was supported by the Council of Scientific and Industrial Research, New‐Delhi, India for the Emeritus Scientist’s Project [Sanction No. 21 (0884)/12/EMR‐II].

References

  1. Paul, S., Kundu, A. and Pal, A. (2014). Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing. J Integr Plant Biol 56(1): 15-23.

简介

小RNA如微小RNA(miRNA),小干扰RNA(siRNA)和其他非编码RNA(包括snRNA和snoRNA)对真核基因调控具有巨大的影响。 高质量小RNA的提取是miRNA的实验分析的重要先决条件。 这将防止RNA降解和去除相关的污染物,包括多酚,多糖和其他次生代谢物。 在这个协议中,我们描述了一种简单的方法,从猕猴桃的叶组织中分离小RNAs,结合两个商业上可用的试剂盒的协议与一些修改。

关键字:microRNA, mymiv, 下一代基因测序, 豇豆蒙哥

材料和试剂

  1. Vigna mungo MYMIV抗性重组近交系,VMR84
  2. mirPremier微小RNA分离试剂盒(Sigma-Aldrich,目录号:SNC10)
  3. Mir-X miRNA FirstStrand合成和SYBR qRT-PCR试剂盒(Takara Bio Company,Clontech,目录号:638314)
  4. 2-巯基乙醇(Sisco Research Laboratories,目录号:1324196)
  5. 乙醇(Merck,目录号:K41540783)
  6. 液氮和干冰
  7. 无RNA酶水(Bangalore Genei,目录号:612151181001730)
  8. 琼脂糖(Sisco Research Laboratories,目录号:0140229)
  9. 溴化乙锭(Sisco Research Laboratories,目录号:054817)
  10. 1x TAE缓冲区(请参阅配方)
  11. 溴化乙锭原料(见配方)

设备

  1. 热循环仪(DNA Engine Cycler,型号:PTC-200)
  2. 实时qPCR(Biorad iQ5实时PCR检测系统)
  3. 离心机(Thermo Fisher Scientific,型号:MicroCL21)
  4. 电泳装置(Bangalore Genei)
  5. 热块或水浴
  6. 砂浆和杵
  7. NanoDrop 1000分光光度计(Thermo Fisher Scientific)

程序

  1. 植物材料和生长条件
    < em> Vigna mungo 重组近交系VMR84用于分离小RNA。
    1. 将成熟的种子用0.1%HgCl 2表面灭菌10分钟,并在去离子水中漂洗两次。
    2. 表面灭菌的种子在潮湿的滤纸中发芽 在28±1℃和70%相对湿度,16小时光照和8小时黑暗
    3. 然后发芽的种子(孵育三天后) 转移到无菌土壤混合物中并在温室中在25±1下生长 C。 在21天的幼苗生长后(在16小时光照和8小时黑暗),幼小   收集三叶叶,在液氮中冷冻并储存 在-80℃直至RNA分离。

  2. 小RNA分离
    1. 使用mirPremier从V豆mungo组织中分离小RNA 微RNA分离试剂盒按照制造商的说明书进行 一些修改。
    2. 使用研钵和杵将收获的叶组织在液氮中研磨成细粉。
    3. 将750μl裂解缓冲液加入到冷冻的组织粉末中(80   mg),涡旋2分钟并在55℃下温育5分钟
    4. 然后将样品以最大速度离心5分钟以除去细胞碎片,基因组DNA和大RNA。
    5. 将溶胞产物上清液(800μl大约)过滤通过 过滤柱,将流出液与100%乙醇混合 立即混合。
    6. 然后使裂解物通过结合 柱,并以最大速度离心30秒, 流出液体倾析。
    7. 结合后,柱子 首先用700μl100%乙醇洗涤,并以14,000×g离心30秒,然后弃去流出物。
    8. 第二 通过向柱中加入500μl结合溶液进行洗涤 以最大速度(14,000×g )离心1分钟
    9. 后来   将500ml乙醇稀释的洗涤溶液2加入到柱中 用于第三次洗涤。 在以最大速度(14,000×g/g)离心后   30秒,弃去流出液。
    10. 进行用乙醇稀释的洗涤溶液2(500ml)的第二次洗涤,并弃去流出液。
    11. 接下来,通过以最大速度(14,000x)离心来干燥柱   g )1分钟。 小心地从柱中取出柱管组件 离心以避免残留的流通液体飞溅   干燥柱。
    12. 使用50ml从柱上洗脱小RNA   洗脱溶液并通过在16,000×g离心和该过程 以提高小RNA产率。 纯化的RNA储存于37℃ -20℃。
    13. 进行了RNA的定量和定性分析 通过NanoDrop用1μl样品,随后是琼脂糖凝胶 电泳分离。 在260至280nm的吸光度比, 通过(A sub-A sub-320)/(A sub-280-Sub 320)计算出的值通常在1.8 和 2.2。

  3. 第一链cDNA合成
    将小RNA多聚腺苷酸化并使用Mir-X miRNA First-Strand Synthesis试剂盒根据制造商的说明书逆转录。
    1. 简言之,将5μlmRQ缓冲液(2x),5μgRNA和1.25μlmRQ酶混合   在10μl的反应体积中,并在热循环仪中温育1小时 在37℃下,然后在85℃终止5分钟以使酶失活。
    2. 逆转录后,通过加入90μl稀释cDNA 核酸酶游离水使总体积达到100μl。 反之 转录的cDNA现在准备用于miRNA定量。

  4. qPCR对miRNA的定量
    使用Mir-X miRNA qPCR SYBR试剂盒进行定量实时PCR。 方法如下:
    1. qPCR引物设计:建议使用整个引物 所需或相关植物物种的成熟miRNA的序列(可获得 在mirBase中)作为miRNA特异性,5'引物。 qPCR的3'引物是 mRQ 3'引物
    2. 通过qPCR扩增miRNA
      1. 使用Δ-δCt方法来量化每个的存在 miRNA相对于U6 snRNA的水平,内部对照。 为了这 实验,对每个cDNA进行U6 snRNA的qPCR扩增 样品重复。 此外,包括适当的 进行每个引物组的模板对照(NTC)以证实 不存在基因组DNA污染。
      2. 对于每个qPCR反应,   μlPCR反应混合物,包含1x SYBR优点 预混物,1×ROX染料,0.2mM的正向和反向引物和50ng   的第一链cDNA。 对于U6反应,正向和反向 使用试剂盒提供的U6引物
      3. qPCR反应 在96孔板中在95℃孵育2分钟,然后是40℃ 95℃10秒和60℃20秒的循环。 扩增循环 随后进行56至95℃的熔解曲线分析   0.5℃,每10秒升温。 每个的熔解曲线 仔细观察扩增子以确认其特异性 引物。记录阈值循环(Ct)值。
    3. 使用Delta-Delta Ct(或ddCt)方法进行数据分析和计算miRNA水平 ddCt方法提供了miRNA的相对水平的测量 通过将它们与归一化标准比较来确定。这里 对每个样品扩增miRNA和U6RNA以确定 Ct值。这允许使用ddCt确定相对水平 计算。
      相对拷贝数使用ddCt方法计算:
      [miR-X] /[miR-X] :2 )
      然而,这种量化方法的精度取决于 引物对的扩增效率应该接近100% 否则量化将不准确。


      图1.熔体曲线 分析在qPCR分析期间产生的mir156。 图表显示了一个图表   的荧光对温度(°C)的负导数 miRNA扩增。 单峰的存在表示非特异性 扩增单一产物。

食谱

  1. 1x TAE(1 L)
    加入242g Tris碱(MW 121.1),57.1ml冰醋酸100ml 0.5M EDTA,并在600ml ddH 2 O中混合,并将最终体积调节至1L。< br/> 然后将20ml50×TAE加入到980ml ddH 2 O中以制备1L 1x TAE(在25℃下pH 8.3)。
  2. 溴化乙锭产品
    将1g溴化乙锭溶解在100ml ddH 2 O中以制备10mg/ml的储备液。

致谢

该协议的原始版本在Paul等人中描述。 (2014年)。 这项工作得到印度新德里科学和工业研究理事会荣誉名誉科学家项目[第21(0884)/12/EMR-II号]的支持。

参考文献

  1. Paul,S.,Kundu,A。和Pal,A。(2014)。 来自叶小RNA的<ν> Vigna mungo 微RNA的鉴定和表达谱 通过深度测序进行转录组。 J Integr Plant Biol 56(1):15-23。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Paul, S., Kundu, A. and Pal, A. (2015). Extraction of Small RNA and qPCR Validation of miRNAs in Vigna mungo. Bio-protocol 5(5): e1417. DOI: 10.21769/BioProtoc.1417.
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