Northern Blot of tRNA in Yeast

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The EMBO Journal
Aug 2009



tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, metabolic labeling can only examine changes in total tRNA, but cannot distinguish different types of tRNAs. The following protocol describes a method to examine individual tRNA gene transcription by northern blot.

Keywords: TRNA (tRNA), Northern Blot (Northern印迹), Yeast (酵母), Urea P (尿素P), TRNA (tRNA), Northern Blot (Northern印迹), Yeast (酵母), Urea PAGE (尿素页), Small RNA (小核糖核酸)

Materials and Reagents

  1. RapidGel (500 ml) (Affymetrix, catalog number: 75848 )
  2. Urea (CO(NH2)2) (Sigma-Aldrich, catalog number: U6504 )
  3. Tetramethylethylenediamine (TEMED, C6H16N2) (Thermo Fisher Scientific, catalog number: 110-18-9 )
  4. APS/ Ammonium persulfate ((NH4)2S2O8) (Sigma-Aldrich, catalog number: A3678 )
  5. Formamide (CH3NO) (Thermo Fisher Scientific, catalog number: 75-12-7 )
  6. DEPC/ Diethylpyrocarbonate (O(COOC2H5)2) (Sigma-Aldrich, catalog number: D5758 )
  7. Bromophenol Blue (C19H10Br4O5S) (Sigma-Aldrich, catalog number: B0126 )
  8. Xylene Cyanol FF (C25H27N2NaO6S2) (Sigma-Aldrich, catalog number: X4126 )
  9. Probe DNA oligo
  10. [γ-32P]-ATP, 10 Ci/mmol 2 mCi/ml, 250 μCi (PerkinElmer, catalog number: BLU002250UC )
  11. Whatman 3 M paper (Whatman, catalog number: 3 MM Chr )
  12. Microspin G-25 column (GE Healthcare, catalog number: 27-5325-01 )
  13. Hybond N+ membrane (Amersham, catalog number: RPN303B )
  14. Ficoll (Sigma-Aldrich, catalog number: F4375 )
  15. Sodium citrate
  16. BSA
  17. Polyvinlypyrrolidone (Sigma-Aldrich, catalog number: PVP360 )
  18. T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201S )
  19. 10x PNK buffer (New England Biolabs, catalog number:  B0201S )
  20. Tris base (Thermo Fisher Scientific, catalog number: 77-86-1 )
  21. Boric acid (Thermo Fisher Scientific, catalog number: 10043-35-3 )
  22. EDTA (Sigma-Aldrich, catalog number: EDS-1KG )
  23. Methylene blue (Sigma-Aldrich, catalog number: 28514-100G )
  24. Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
  25. Salmon sperm DNA (Life Technologies, InvitrogenTM, catalog number: 15632-011 )


  1. Bench top centrifuge
  2. UV cross linker (Strata linker,model: 1800 )
  3. Power supply with constant voltage > 450 V
  4. Exposure cassette / intensifier screen (Sigma-Aldrich, catalog number: C5479-1EA )
  5. Hybridization bottle
  6. Hybridization oven
  7. Large gel cassette (around 20 x 40 cm)
  8. Film developer


Part I: Separate tRNAs by denaturing gel

  1. Prepare total RNA by either Trizol RNA extraction kit or by hot phenol method described in Wei (2012). Good quality RNA will have an OD260/280 ratio of 1.8 to 2 and an OD260/230 of 1.8 or greater.
  2. Prepare 10% Acrylamide-6 M Urea denaturing gel in a large gel cassette (around 20 x 30 cm. Mini gel did not work well in my experiment).
  3. Pre-run the gel in 1x TBE buffer with constant 450 V until the gel is heated to 50 °C (about 1 h). Note: I found this to be critical. One reason could be that pre-running the gel to this temperature could help get rid of excessive Urea in the gel, making RNA possible to go through. I usually attached a thermometer to ensure that the temperature reach 50 °C.
  4. RNA samples are mixed with 2x with Formamide loading dye and heated to 70 °C for 2 min.
  5. Turn off power. Rinse out the wells with 1x TBE using a syringe and needle, make sure that urea is rinsed out.
  6. Load the RNA samples (25 μg) and run the gel at constant 450 V for about 2 h (Bromophenol Blue runs around 12 nt and cyanol around 55 nt).
  7. Let the gel cool down to room temperature and disassemble the gel set.

Part II: transfer tRNAs to membrane

  1. Measure gel and cut a piece of Hybond N+ membrane with clean gloves and scissors (washed with DEPC water and autoclaved). Soak the membrane in 1x TBE, attach to the gel. Soak 2 pieces of autoclaved Whatman 3 M paper in 1x TBE and sandwich the gel-membrane.
  2. Transfer gel/membrane/whaman paper sandwich to semi-dry transfer apparatus. Use a sterile transfer pipet to squeeze out bubbles between the membrane and the gel.
  3. Wipe off excess liquid around the edges of gel and transfer at constant 10 V for 2.5 h.
  4. Disassemble the gel and membrane, soak membrane in DEPC water for 1 min. Put the membrane on autoclaved filter paper for 10 min to dry the membrane.
  5. Crosslink the RNAs to the membrane with UV cross linker (Strata Linker 1800) at 1,200 x 100 m Joles. It takes roughly 2 min.
  6. Stain with methylene blue RNA staining solution for 1 min. De-stain by washing with DEPC water several times for 30 sec. This is to evaluate the efficiency of RNA transfer. If transfer is not good, I suggest repeating the above procedures since radioactive material is involved in later steps.
  7. Save the membrane in DEPC water for Northern blotting.

Part III: 5' End-Labeling of Probe

  1. To examine the newly synthesized tRNA, design DNA oligo annealing specifically to the pre-tRNA rather than mature tRNA, because mature RNA is very abundant. Pre-tRNAs will be processed to mature tRNA. During this process, certain DNA sequence will be cleaved. Primers will be designed annealing to the cleaved DNA sequence For example, pre-tRNA Leu3 probe is 5'-CCAAACAACCACTTATTTGTTGA-3'.
  2. Prepare 5' end labeling reaction buffer and label the probe with radioactive ATP at 37 °C for 1 h (see Recipes for detail).
  3. Prepare the microspin G-25 column by vortexing the resin in the column.
  4. Snap off bottom and loosen the cap, place in 1.5 ml Eppendorf tubes and spin the column for 1 min at 700 x g. The gentle centrifugation is critical for the column to form a smooth angled surface.
  5. Place the column in new Eppendorf tube and slowly apply the labeling reaction to center of angled surface of resin bed. Do not disturb the resin.
  6. Spin column for 2 min at 700 x g. Discard column in radioactive waste.
  7. Take out 1 μl of labeled probe in 0.5 ml eppendorf tube, put tube in scintillation vial and count the cpm.

Part IV: Hybridization

  1. Mix hybridization buffer and take 20 ml into 50 ml conical tube.
  2. Heat the buffer in 65 °C water bath until clear. It should be clear within 10 min.
  3. Transfer 10 ml to hybridization bottle with membrane (note transferred side is the proved side) and rotate gently at 37 °C in a hybridization oven for 60 min.
  4. Put the remaining 10 ml hybridization at 37 °C for 30 min.
  5. Heat 100 μl salmon sperm DNA at 90 °C for 1 min and add to the remaining 10 ml hybridization buffer.
  6. Add 20 μl labeled probe into the 10 ml hybridization buffer and shake vigorously.
  7. Decant the buffer in the hybridization bottle and add the new hybridization buffer with labeled probe. Rotate gently at 37 °C in a hybridization oven overnight.
  8. Pre-warm wash buffer to 37 °C in a water bath.
  9. Transfer hybridization buffer with probes to 50 ml conical tube and store it at -20 °C. The probes can be re-used within 2 weeks for 2-3 times, but the auto-radioactive signal will be decreasing.
  10. Wash the membrane with 50 ml wash buffer by rotating at 42 °C for 30 min in the hybridization oven.
  11. Repeat washing for 2 more times.
  12. Take out membrane and wrap with Saran-wrap.
  13. Place the membrane in exposure cassette with intensifier screen and film, put at -80 °C refrigerator.
  14. Develop the film after 3 days. If signal is weak, expose for 1 week.


  1. DEPC water
    Add 1 ml DEPC to 1 L ddH2O2, mix and put at room temperature overnight. Autoclave.
  2. 10x TBE in DEPC water
    In 800 ml DEPC water, add 108 g Tris base, 55 g boric acid, 40 ml of 0.5 M EDTA (pH 8.0).
    Mix to dissolve and add DEPC water to 1L.
  3. 10% Acrylamide-6 M Urea denaturing gel
    1. Mix 2.5 ml 10x TBE, 6.25 ml Rapid Gel (40%) and 15 g Urea, heat to 50 °C and mix to dissolve.
    2. Add DEPC water to 25 ml then filter through 0.45 μM filter syringe.
    3. Add 25 μl TEMED and 50 μl 25% APS, mix vigorously and transfer to large gel set with appropriate comb.
  4. 2x Formamide loading dye
    95% (v/v) formamide in DEPC water, add tiny amount of Bromophenol Blue (0.01~0.1%) and Xylene Cyanol FF (0.01~0.1%) , vortex to mix.
  5. Methylene blue RNA staining solution
    Dissolve sodium acetate in water to 0.3 M and adjust to pH 5.5 with glacial acetic acid and NaOH. Dissolve 0.02 g methylene blue in 100 ml of above solution and filtered.
  6. 5' end labeling reaction buffer
    2 μl
    10x PNK buffer (fresh from NEB)                                
    30 pmol
    probe DNA oligo
    3.3 μl
    [γ-32P] ATP
    1 μl
    T4 Polynucleotide Kinase 10 U/ml
    Add DEPC H2O to 20 μl
    Incubate at 37 °C at least 1 h.
  7. 20X SSC (1 L)
    NaCl 175 g
    Sodium citrate 88 g
    Add DEPC water to 1 L and autoclave
  8. 50x Denhardt's Solution (100 ml)
    1 g                          
    1 g
    1 g
    Add DEPC water to 100 ml to dissolve, filter solution through 0.45 μm filter syringe.
  9. Hybridization buffer (1 L)
    250 ml 
    20x SSC          
    100 ml
    Denhart's reagent (50x)
    10 ml
    10% SDS (autoclaved)
    Add DEPC water to 1 L, store in 4 °C
  10. Wash buffer (1 L)
    100 ml 20x SSC
    10 ml 10% SDS (autoclaved)
    Add 890 ml DEPC water and store at room temperature.


This protocol is derived from the following papers, Wei et al. (2009a) and Wei et al. (2009b), and the relevant references therein. The work was supported by NIH grants R01-CA099004 and R01-CA123391 to Dr. X.F. Steven Zheng at Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey.


  1. Wei, Y., Tsang, C. K. and Zheng, X. F. (2009a). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28(15): 2220-2230.
  2. Wei, Y. and Zheng, X. F. (2009b). Sch9 partially mediates TORC1 signaling to control ribosomal RNA synthesis. Cell Cycle 8(24): 4085-4090.
  3. Wei, Y. (2012). A simple preparation of RNA from yeast by hot phenol for northern blot. Bio-protocol 2(9): e209. 


tRNA是约70-90nt的小RNA。 tRNA与许多其他小RNA不同,因为它们是非常丰富的,这使得难以通过常规Northern印迹研究它们的转录调节。 传统的Northern印迹涉及通过聚合掺入放射性核苷酸,然而,tRNA对于聚合来说太短。 传统的Northern印迹检测RNA水平的变化,然而,tRNA是如此丰富,其水平的小变化将逃脱检测。 由于这些原因,已经使用了放射性尿嘧啶的代谢标记。 然而,代谢标记只能检查总tRNA的变化,但不能区分不同类型的tRNA。 以下方案描述了通过Northern印迹检查单个tRNA基因转录的方法。

关键字:tRNA, Northern印迹, 酵母, 尿素P, tRNA, Northern印迹, 酵母, 尿素页, 小核糖核酸


  1. RapidGel(500ml)(Affymetrix,目录号:75848)
  2. 尿素(CO(NH 2)2)(Sigma-Aldrich,目录号:U6504)
  3. 四甲基乙二胺(TEMED,C 6 H 16 N 2)(Thermo Fisher Scientific,目录号:110-18-9)
  4. APS /过硫酸铵((NH 4)2 S 2 S 2 O 8)(Sigma-Aldrich,目录号 :A3678)
  5. 甲酰胺(CH 3 NO)(Thermo Fisher Scientific,目录号:75-12-7)
  6. DEPC /焦碳酸二乙酯(O(COOC H 2 H 5)2)(Sigma-Aldrich,目录号:D5758)
  7. 溴代酚蓝(C 19 H 10 BrB 4 O 5 S)(Sigma-Aldrich,目录号:B0126 )
  8. 二甲苯Cyanol FF(C 25 H 27 N 2nd N 2 NaO 6 S sub 2) (Sigma-Aldrich,目录号:X4126)
  9. 探针DNA寡聚体
  10. [γ-sup 32 P] -ATP,10Ci/mmol 2mCi/ml,250μCi(PerkinElmer,目录号:BLU002250UC)
  11. Whatman 3 M纸(Whatman,目录号:3 MM Chr)
  12. Microspin G-25柱(GE Healthcare,目录号:27-5325-01)
  13. Hybond N +膜(Amersham,目录号:RPN303B)
  14. Ficoll(Sigma-Aldrich,目录号:F4375)
  15. 柠檬酸钠
  16. BSA
  17. 聚乙烯吡咯烷酮(Sigma-Aldrich,目录号:PVP360)
  18. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  19. 10x PNK缓冲液(New England Biolabs,目录号:B0201S)
  20. Tris碱(Thermo Fisher Scientific,目录号:77-86-1)
  21. 硼酸(Thermo Fisher Scientific,目录号:10043-35-3)
  22. EDTA(Sigma-Aldrich,目录号:EDS-1KG)
  23. 亚甲基蓝(Sigma-Aldrich,目录号:28514-100G)
  24. 乙酸钠(Sigma-Aldrich,目录号:S2889)
  25. 鲑精DNA(Life Technologies,Invitrogen TM,目录号:15632-011)


  1. 台式离心机
  2. UV交联剂(Strata接头,型号:1800)
  3. 恒压电源> 450 V
  4. 曝光盒/增强屏(Sigma-Aldrich,目录号:C5479-1EA)
  5. 杂交瓶
  6. 杂交炉
  7. 大凝胶盒(约20×40厘米)
  8. 电影开发商



  1. 通过Trizol RNA提取试剂盒或通过Wei(2012)中描述的热酚法制备总RNA。优质RNA将具有1.8至2的OD比260/280比和1.8或更大的OD 260/230比。
  2. 在大凝胶盒(大约20×30厘米。迷你凝胶在我的实验中不能很好地工作)中制备10%丙烯酰胺-6M尿素变性凝胶。
  3. 在1×TBE缓冲液中以恒定450V预运行凝胶,直到凝胶加热至50℃(约1小时)。 注意:我发现这是至关重要的。一个原因可能是将凝胶预先运行到这个温度可以帮助除去凝胶中过多的尿素,使得RNA可能通过。我通常连接一个温度计,以确保温度达到50℃。
  4. RNA样品与2x与甲酰胺负载染料混合,并加热至70℃2分钟
  5. 关闭电源。使用注射器和针头用1x TBE冲洗孔,确保尿素被冲洗掉
  6. 加载RNA样品(25μg),并在恒定的450V下运行凝胶约2小时(溴酚蓝运行约12nt,cyanol运行约55nt)。
  7. 让凝胶冷却至室温,拆开凝胶组


  1. 测量凝胶并用干净的手套和剪刀(用DEPC水洗涤并高压灭菌)切割一块Hybond N +膜。 将膜浸泡在1×TBE中,附着到凝胶上。 浸泡2件高压灭菌的Whatman 3 M纸在1 TBE和夹层凝胶膜。
  2. 转移凝胶/膜/whaman纸夹层到半干式转移装置。 使用无菌输送吸管挤出膜和凝胶之间的气泡
  3. 擦去凝胶边缘周围的多余液体,并在恒定的10 V下转移2.5小时
  4. 拆解凝胶和膜,浸泡膜在DEPC水中1分钟。 将膜置于高压灭菌的滤纸上10分钟以干燥膜。
  5. 用UV交联剂(Strata Linker 1800)以1,200×100m Joles将RNA交联到膜上。 大约需要2分钟。
  6. 用亚甲蓝RNA染色溶液染色1分钟。 通过用DEPC水洗涤数次来脱色30秒。 这是为了评估RNA转移的效率。 如果转移不好,我建议重复上述程序,因为放射性物质参与后面的步骤。
  7. 将膜保存在DEPC水中用于Northern印迹


  1. 为了检查新合成的tRNA,设计特异性针对pre-tRNA而不是成熟tRNA的DNA寡核苷酸,因为成熟RNA是非常丰富的。 前tRNA将被加工成成熟tRNA。 在该过程中,某些DNA序列将被切割。 引物将被设计为与切割的DNA序列退火。例如,pre-tRNA Leu3探针是5'-CCAAACAACCACTTATTTGTTGA-3'。
  2. 准备5'端标记反应缓冲液,并用放射性ATP在37°C标记探针1小时(详见配方详细信息)。
  3. 通过旋转柱中的树脂制备微球蛋白G-25柱
  4. 扣下底部并松开帽,置于1.5ml Eppendorf管中,并以700xg旋转柱1分钟。 温和的离心对于柱形成平滑的成角度的表面是至关重要的
  5. 将柱置于新的Eppendorf管中,并缓慢地将标记反应应用于树脂床的成角度表面的中心。 请勿打扰树脂。
  6. 在700×g下旋转柱2分钟。 废弃放射性废物中的色谱柱。
  7. 取出1微升的标记探针在0.5毫升eppendorf管,将管放入闪烁瓶和计数cpm。


  1. 混合杂交缓冲液,并取20ml到50ml锥形管中。
  2. 将缓冲液在65℃水浴中加热至澄清。 应在10分钟内清除。
  3. 转移10毫升到带有膜(钞票转移侧是证明的一面)的杂交瓶,并在杂交炉中在37℃轻轻旋转60分钟。
  4. 将剩余的10ml杂交在37℃下放置30分钟
  5. 加热100微升鲑鱼精子DNA在90℃下1分钟,加入剩余的10毫升杂交缓冲液。
  6. 将20μl标记的探针加入到10 ml杂交缓冲液中,并剧烈摇动
  7. 滗析杂交瓶中的缓冲液,并加入带有标记探针的新杂交缓冲液。 在杂交烘箱中在37℃轻轻旋转过夜。
  8. 将预温热的洗涤缓冲液在水浴中37℃。
  9. 转移杂交缓冲液与探头到50毫升锥形管,并将其存储在-20°C。 探针可在2周内重复使用2-3次,但自动放射性信号会减少
  10. 通过在42℃下在杂交炉中旋转30分钟,用50ml洗涤缓冲液洗涤膜
  11. 重复洗涤2次以上。
  12. 取出膜并用Saran包装包裹。
  13. 将膜置于带增感屏和胶片的曝光盒中,置于-80℃冰箱中
  14. 3天后开发电影。 如果信号弱,暴露1周。


  1. DEPC水
    将1ml DEPC加入1L ddH 2 O 2中,混合并在室温下放置过夜。 高压灭菌。
  2. DEPC水中10x TBE
    在800ml DEPC水中,加入108g Tris碱,55g硼酸,40ml 0.5M EDTA(pH 8.0)。
    混合溶解并加入DEPC水至1L 高压灭菌。
  3. 10%丙烯酰胺-6M尿素变性凝胶
    1. 将2.5ml 10×TBE,6.25ml快速凝胶(40%)和15g尿素混合,加热至50℃并混合溶解。
    2. 加入DEPC水至25ml,然后通过0.45μM过滤器注射器过滤
    3. 加入25微升TEMED和50微升25%APS,剧烈混合,并转移到大凝胶设置与适当的梳子。
  4. 2x甲酰胺负载染料
    95%(v/v)甲酰胺的DEPC水溶液中,加入微量的溴酚蓝(0.01〜0.1%)和二甲苯Cyanol FF(0.01〜0.1%
  5. 亚甲蓝RNA染色溶液
    将乙酸钠在水中溶解至0.3M,并用冰醋酸和NaOH调节至pH5.5。 将0.02g亚甲基蓝溶解在100ml上述溶液中并过滤。
  6. 5'端标记反应缓冲液
    10x PNK缓冲区(新鲜来自NEB)                              
    30 pmol
    [γ- 32 P] ATP
    T4多核苷酸激酶10 U/ml
    将DEPC H sub 2 O加到20μl
    中 在37℃孵育至少1小时。
  7. 20X SSC(1L)
    NaCl 175g
  8. 50x Denhardt's溶液(100ml)
    1 g                           ; 
  9. 杂交缓冲液(1 L)
    250 ml 
    20x SSC           
    100 ml
    10 ml
    将DEPC水加入1 L,在4℃下保存
  10. 洗涤缓冲液(1 L)
    100ml 20×SSC
    10ml 10%SDS(高压灭菌) 加入890ml DEPC水并在室温下储存


该协议衍生自以下论文:Wei等人(2009a)和Wei等人(2009b)及其中的相关参考文献。 该工作由NIH授予R01-CA099004和R01-CA123391支持给Dr. X.F. Steven Zheng在Robert Wood Johnson医学院,新泽西州医学和牙科大学。


  1. Wei,Y.,Tsang,C.K。和Zheng,X.F。(2009a)。 TORC1调节RNA聚合酶III依赖性转录的机制。 EMBO J 28(15):2220-2230
  2. Wei,Y。和Zheng,X.F。(2009b)。 Sch9部分介导TORC1信号转导以控制核糖体RNA合成。 Cell Cycle 8(24):4085-4090。
  3. Wei,Y。(2012)。 通过热苯酚用于Northern印迹的来自酵母的RNA的简单制备。 生物方案 2(9):e209。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wei, Y. (2013). Northern Blot of tRNA in Yeast. Bio-protocol 3(7): e464. DOI: 10.21769/BioProtoc.464.