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RNA Isolation and Northern Blot Analysis
RNA分离和Northern印迹杂交分析   

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Abstract

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression profiles by northern blot in Vero cells and H1299 cells after IBV infection. RNA was extracted in IBV (infectious bronchitis virus) infected cells and electrophoresis was used to separate the RNA sample. RNA was transferred from the electrophoresis gel to the blotting membrane by capillary transfer. Specific mRNA was detected with hybridization probes complementary to part of target sequence. The probes were prepared by RT-PCR and labeled by digoxigenin (DIG) using DIG labeling kit.

Materials and Reagents

  1. Vero cells (kidney epithelial cells extracted from an African green monkey) (ATCC, catalog number: CCL-81 TM)
  2. H1299 cells (human lung carcinoma cell line) (ATCC, catalog number: CRL-5803 TM)
  3. The egg-adapted Beaudette strain of IBV (ATCC, catalog number: VR-22 )
  4. Dulbecco modified Eagle medium (DMEM) (Life Technologies, Gibco®, catalog number: 11960-044 )
    Note: It contains more vitamins and more glucose, as well as iron and is suitable for most types of cells.
  5. Roswell Park Memorial Institute medium (RPMI) 1640 (Life Technologies, Gibco®, catalog number: 21870-076 )
    Note: This medium contains a great deal of phosphate and is formulated for use in a 5% carbon dioxide atmosphere.
  6. Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 25200-072 )
  7. TRIzol reagent (Life Technologies, Gibco®, catalog number: 15596-018 )
  8. Chloroform (Thermo Fisher Scientific, catalog number: C/4960/17 )
  9. Isopropanol (Thermo Fisher Scientific, catalog number: P/7507/17 )
  10. Ethanol (Merck KGaA, catalog number: 1.00983.2500 )
  11. RNase free water
  12. Reverse transcriptase AMV (Roche Diagnostics, catalog number: 10109118001 )
  13. Oligo (dT) (1st Base Biochemicals)
  14. RNasin® ribonuclease inhibitor (Promega Corporation, catalog number: N2511 )
  15. Primers (1st Base Biochemicals)
  16. DIG labeling kit (Roche, catalog number: 11175025910 )
  17. RNA loading buffer (New England Biolabs, catalog number: B0363S )
  18. Agarose (1st Base Biochemicals, catalog number: BIO-100-500G )
  19. Formaldehyde (Thermo Fisher Scientific, catalog number: F75P1GAL ))
  20. Ethidium bromide (Bio-Rad Laboratories, catalog number: 1610433 )
  21. HybondTM-N+ membrane (Amersham Biosciences, catalog number: RPN303B )
  22. DIG Wash and Block Buffer Set (Roche Diagnostics, catalog number: 11585762001 )
  23. DIG easy Hyb (Roche Diagnostics, catalog number: 11603558001 )
  24. Anti-digoxigenin-AP fab fragments (Roche Diagnostics, catalog number: 11093274910 )
  25. CDP-Star (Roche Diagnostics, catalog number: 12041677001 )
  26. Amersham hyperfilm ECL (Amersham Biosciences, catalog number: 28906837 )
  27. 70% RNase-free ethanol
  28. Tris(hydroxymethyl)aminomethane (Tris base) (Promega Corporation, catalog number: H5135 )
  29. Acetic acid (Glacial) (Merck KGaA, catalog number: 1.00063.2500 )
  30. 3-(4-morpholino) propane sulfonic acid (MOPS) (Thermo Fisher Scientific, catalog number: BP308-500 )
  31. Sodium acetate.3H2O (Thermo Fisher Scientific, catalog number: S207-10 )
  32. Sodium Citrate (Thermo Fisher Scientific, catalog number: S25545 )
  33. 10x TAE Electrophoresis Buffer (1 L) (see Recipes)
  34. 10x MOPS buffer (1 L) (see Recipes)
  35. 1x MOPS buffer (1 L) (see Recipes)
  36. 1.3% Formaldehyde Agarose gel (see Recipes)
  37. 20x SSC buffer (1 L) (see Recipes)
  38. 2x SSC, 0.1% SDS (1 L) (see Recipes)
  39. 0.1x SSC, 0.1% SDS (see Recipes)

Equipment

  1. 100 mm cell culture dishes (Corning, catalog number: 430167 )
  2. 0.2 ml thin-wall Gene-Amp PCR tube (Corning, Axygen®, catalog number: PCR-02-C )
  3. FormaTM Steri-CycleTM CO2 Incubators (Thermo Fisher Scientific, catalog number: 201370 )
  4. OLYMPUS CKX31 microscope
  5. Eppendorf centrifuge 5415R
  6. NanoDrop (Thermo Fisher Scientific, model: ND-1000 spectrophotometer )
  7. Power Pac and electrophoresis tank (Bio-Rad Laboratories)
  8. Tray
  9. Glass plate
  10. Tissue paper
  11. CL-1000, ultraviolet crosslinker (UVP)
  12. Hybaid Maxi 14 Hybridization Oven (Thermo Fisher Scientific)
  13. Hybridization tubes
  14. Kodak Biomax cassette (Eastman Kodak Company)
  15. Kodak X-OMAT 2000 processor (Eastman Kodak Company)

Procedure

  1. RNA extraction
    1. Cells were seeded in 100-mm-diameter dishes and infected with either 2 PFU of live IBV per cell or the same amount of UV-inactivated IBV (UV-IBV) at 37 °C. Excess virus in the medium was removed by replacing with fresh medium at 1 h post-infection.
    2. The IBV-infected cells were incubated at 37 °C in 5% CO2.
    3. At the indicated time points (0, 2, 4, 8, 12, 16, 20, 24, 28 h post-infection), cells were rinsed with 10 ml Phosphate Buffered Saline (PBS) buffer once and lysed in 1 ml TRIzol for 5 min at room temperature.
    4. Cell lysates were transfer into eppendorf tubes and one-fifth (volume/volume) of chloroform was added to each tube.
    5. Shake tubes vigorously by hand for 15 sec and incubated for 3 min at room temperature, then centrifuged at 12,000 x g for 15 min at 4 °C.
    6. The upper aqueous phase was transfer into a new tube and mixed with 1:1 (volume/volume) of 100% isopropanol, and then incubated for 10 min at room temperature.
    7. RNA was precipitated by centrifugation at 12,000 x g for 10 min at 4 °C.
    8. RNA pellet was washed with 1 ml 70% RNase-free ethanol once and spin down by 7,500 x g for 5 min.
    9. The RNA pellets are air-dried and dissolved in 100 µl RNase-free H2O by incubating at 65 °C for 15 min.
    10. RNA concentration and purity were determined by NanoDrop.
    11. The RNAs were stored at -80 °C for further use.

  2. Probe preparation
    1. Northern blot probes were obtained by RT-PCR and labeled by digoxigenin (DIG) using DIG labeling kit described as follow steps.
    2. 2 µg of total RNA is added to 2 µl of 10 pmoles of an oligo (dT) in a sterile 0.2 ml thin-wall Gene-Amp PCR tube of a final volume of 10.5 µl.
    3. After denaturation at 65 °C for 10 min, the tubes are cooled on ice immediately.
    4. The denatured RNA-primer mixture is then added to a final volume of 20 µl reaction mixture containing 10 mM of dNTPs, 20 units of Rnasinâ ribonuclease inhibitor, 1x Expandä reverse transcriptase buffer and 50 units of reverse transcriptase.
    5. The first strand cDNA is synthesized at 43 °C for 1 h, and reaction can be terminated by heating at 65 °C for 10 min (optional).
    6. Amplification of cDNA was achieved by polymerase chain reaction (PCR) in a 25 or 50 µl reactions containing of appropriate primer pairs and PFU polymerase using the DIG labeling kit according to the manufacturer’s manual.
    7. Primers used for human ATF4 were 5’-CCGTCCCAAACCTTACGATC-3’ (forward) and 5’-ACTATCCTCAACTAGGGGAC-3’ (reverse). Primers used for human ATF3 were 5’-GGTTAGGACTCTCCACTCAA-3 (forward) and 5’-AGACAGTAGCCAGCGTCCTT-3’ (reverse). Primers used for human GADD153 were 5'-GATTCCAGTCAGAGCTCCCT3' (forward) and 5'-GTAGTGTGGCCCAAGTGGGG-3' (reverse). Prepare a 10x concentration solution of each respective PCR primer.
    8. Add the following reagents in a 0.2 ml reaction tube on ice, in the following order: ddH2O 32.25 µl, PCR buffer 5 µl, PCR DIG labeling mix 5 µl, forward primer 5 µl, reverse primer 5 µl, enzyme mix 0.75 µl, template cDNA 2 µl, final volume 50 µl. Vortex the mixture and centrifuge briefly.
    9. Place the sample in a thermal block cycler and perform PCR in following condition: initial denature at 95 °C for 2 min, denature at 95 °C for 10 sec, anneal at 60 °C for 30 sec, and elongate at 72 °C for 2 min, repeat denaturation, annealing, and elongation for 30 cycles, finally elongate at 72 °C for 7 min.
    10. Run a portion of each PCR reaction on an agarose mini gel and then stain the gel with ethidium bromide and examine the PCR products under UV.

  3. Northern blot
    1. To analyze RNA expression by Northern blot, 30 µg of RNA from each sample preparation was mixed with RNA loading buffer and load on wells in 1.3% agarose formaldehyde gel (see Recipes).
    2. Run the gel with 3-4 V/cm in RNase free gel boxes for 4 h until the RNAs are well separated.
    3. Stain the gel briefly in 0.25 – 0.5 µg/ml ethidium bromide and examine the gel under UV light.
    4. Rinse gels for 2 x 15 min in 20x SSC and RNA on the gel were transferred onto a HybondTM-N+ membrane by capillary transfer with 20x SSC overnight at room temperature.
    5. Fix the RNA to the membrane by UV-crosslinking. The energy used is 20,000 µJoules/cm2 at 245 nm.
    6. After the UV-crosslinking, rinse the membranes briefly in ddH2O and allow to air-dry.
    7. Prehybridize membranes with DIG easy Hyb for 30 min with gentle agitation at 68 °C.
    8. Denature DIG-labeled RNA probes by boiling for 5 min and rapidly cooling in ice, and add the denatured probes (25 ng/ml) to 10 ml prewarmed DIG Easy Hyb.
    9. 10 ml probe/hybridization mixtures were added to membranes and incubated for 6 h at 68 °C with gentle agitation.
    10. After hybridization, membranes were washed with 2x SSC, 0.1% SDS for 2 x 5 min at 25 °C under constant agitation, and then washed with 0.1x SSC, 0.1% SDS for 2 x 15 min at 68 °C under constant agitation.
    11. The membranes were then rinsed briefly (5 min) in washing buffer and was blocked in blocking buffer for 30 min.
    12. After blocking, membranes were incubated with DIG antibody (Dilute anti-DIG-AP 1:10,000 in blocking buffer) for 30 min, washed 2 x 15 min in washing buffer and equilibrated 3 min in detection buffer.
    13. The signal was detected with CDP-Star according to the manufacturer’s instructions (see Figures 1-3).


      Figure 1. Northern blot analysis of ATF4 mRNA in IBV-infected cells. Vero and H1299 cells were infected with IBV (MOI~1) and harvested at indicated time points. Total RNA was isolated and subjected to Northern blot using an ATF4 probe. Ethidium bromide staining of 28S rRNA and 18S rRNA is shown as a loading control. Band intensities of ATF4 were determined and normalized to rRNA.


      Figure 2. Northern blot analysis of ATF3 mRNA in IBV-infected cells. Vero and H1299 cells were infected as in Figure 1. RNA extraction and Northern blot was performed as in Figure 1 using ATF3 probe.


      Figure 3. Northern blot analysis of GADD153 at the mRNA level in IBV-infected cells. Vero and H1299 cells were infected with IBV (MOI~1) or incubated with UV-IBV and harvested at indicated time points. RNA extraction and Northern blot was performed as in Figure 1 using GADD153 probe.

Recipes

  1. 10x TAE Electrophoresis Buffer (1 L)
    48.4 g Tris(hydroxymethyl)aminomethane (Tris base)
    11.4 ml 17.4 M glacial acetic acid
    3.7 g EDTA, disodium salt
    ddH2O
  2. 10x MOPS buffer (1 L)
    83.7 g 3-(N-morpholino) propanesulfonic acid (MOPS)
    13.61 g Sodium acetate.3H2O
    3.7 g EDTA
    ddH2O
  3. 1x MOPS buffer (1 L)
    100 ml 10x MOPS buffer
    20 ml 37%-formaldehyde
    880 ml ddH2O
  4. 1.3% Formaldehyde Agarose gel
    1.3 g agarose
    10 ml 10x Formaldehyde Agarose gel buffer
    Add RNase-free water to 100 ml
    Heat the mixture to melt agarose
    Cool to 65°C in a water bath.
    Add 1.8 ml of 37% (12.3 M) formaldehyde (toxic) and 1 µl of a 10 mg/ml ethidium Bromide stock solution
    Mix thoroughly and pour onto gel support
    Prior to running the gel, equilibrate in 1x Formaldehyde Agarose gel running buffer for at least 30 min
  5. 20x SSC buffer (1 L)
    175.3 g of NaCl
    88.2 g of Sodium Citrate
    ddH2O
    Adjust the pH to 7.0 with a few drops of 14 N solution of HCl
    Sterilized by autoclaving
  6. 2x SSC, 0.1% SDS (1 L)
    100 ml 20x SSC buffer
    10 ml 10%SDS
    890 ml ddH2O
  7. 0.1x SSC, 0.1% SDS
    10 ml 20x SSC buffer
    10 ml 10%SDS
    980 ml ddH2O

Acknowledgments

This protocol was adapted from the previous publication Liao et al. (2013). This work was partially supported by a Competitive Research Programme (CRP) grant (R-154-000-529-281) from the National Research Foundation, Singapore.

References

  1. Liao, Y., Fung, T. S., Huang, M., Fang, S. G., Zhong, Y. and Liu, D. X. (2013). Upregulation of CHOP/GADD153 during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway. J Virol 87(14): 8124-8134.

简介

Northern印迹是在分子生物学研究中用于通过检测样品中的RNA来研究基因表达的技术。 使用Northern印迹,可以在分化,形态发生以及异常或疾病状况期间观察到特定的基因表达水平。 在这里,我们检查ATF3,ATF4和GADD153基因表达谱通过北部污点在Vero细胞和H1299细胞后IBV感染。 在IBV(感染性支气管炎病毒)感染的细胞中提取RNA,并使用电泳分离RNA样品。 通过毛细管转移将RNA从电泳凝胶转移到印迹膜上。 使用与靶序列的一部分互补的杂交探针检测特异性mRNA。 通过RT-PCR制备探针,并使用DIG标记试剂盒通过地高辛(DIG)标记。

材料和试剂

  1. Vero细胞(从非洲绿猴提取的肾上皮细胞)(ATCC,目录号:CCL-81 TM
  2. H1299细胞(人肺癌细胞系)(ATCC,目录号:CRL-5803 TM
  3. 适应于IBV的Beaudette菌株(ATCC,目录号:VR-22)
  4. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:11960-044)
    注意:它含有更多的维生素和更多的葡萄糖,以及铁,适合大多数类型的细胞。
  5. Roswell Park Memorial Institute培养基(RPMI)1640(Life Technologies,Gibco ,目录号:21870-076)
    注意:该培养基含有大量的磷酸盐,并配制用于5%二氧化碳气氛。
  6. 胰蛋白酶/EDTA(Life Technologies,Gibco ,目录号:25200-072)
  7. TRIzol试剂(Life Technologies,Gibco ,目录号:15596-018)
  8. 氯仿(Thermo Fisher Scientific,目录号:C/4960/17)
  9. 异丙醇(Thermo Fisher Scientific,目录号:P/7507/17)
  10. 乙醇(Merck KGaA,目录号:1.00983.2500)
  11. 无RNase水
  12. 逆转录酶AMV(Roche Diagnostics,目录号:10109118001)
  13. Oligo(dT)(1 Base Biochemicals)
  14. RNasin核糖核酸酶抑制剂(Promega Corporation,目录号:N2511)
  15. 引物(1 Base Base Biochemicals)
  16. DIG标记试剂盒(Roche,目录号:11175025910)
  17. RNA加载缓冲液(New England Biolabs,目录号:B0363S)
  18. 琼脂糖(1μlBase Biochemicals,目录号:BIO-100-500G)
  19. 甲醛(Thermo Fisher Scientific,目录号:F75P1GAL))
  20. 溴化乙锭(Bio-Rad Laboratories,目录号:1610433)
  21. Hybond TM膜(Amersham Biosciences,目录号:RPN303B)
  22. DIG洗涤和封闭缓冲液组(Roche Diagnostics,目录号:11585762001)
  23. DIG easy Hyb(Roche Diagnostics,目录号:11603558001)
  24. 抗洋地黄毒苷-AP fab片段(Roche Diagnostics,目录号:11093274910)
  25. CDP-星级(Roche Diagnostics,目录号:12041677001)
  26. Amersham hyperfilm ECL(Amersham Biosciences,目录号:28906837)
  27. 70%无RNA酶的乙醇
  28. 三(羟甲基)氨基甲烷(Tris碱)(Promega Corporation,目录号:H5135)
  29. 乙酸(Glacial)(Merck KGaA,目录号:1.00063.2500)
  30. 3-(4-吗啉代)丙烷磺酸(MOPS)(Thermo Fisher Scientific,目录号:BP308-500)
  31. 乙酸钠 3H 2 O(Thermo Fisher Scientific,目录号:S207-10)
  32. 柠檬酸钠(Thermo Fisher Scientific,目录号:S25545)
  33. 10x TAE电泳缓冲液(1 L)(参见配方)
  34. 10x MOPS缓冲液(1 L)(参见配方)
  35. 1x MOPS缓冲液(1 L)(参见配方)
  36. 1.3%甲醛琼脂糖凝胶(见配方)
  37. 乙酸钠 3H 2 O(Thermo Fisher Scientific,目录号:S207-10)
  38. 柠檬酸钠(Thermo Fisher Scientific,目录号:S25545)
  39. 10x TAE电泳缓冲液(1 L)(参见配方)
  40. 10x MOPS缓冲液(1 L)(参见配方)
  41. 1x MOPS缓冲液(1 L)(参见配方)
  42. 1.3%甲醛琼脂糖凝胶(见配方)
  43. ...
  44. FormaTM Steri-CycleTM CO2 Incubators (Thermo Fisher Scientific, catalog number: 201370)
  45. OLYMPUS CKX31 microscope
  46. Eppendorf centrifuge 5415R
  47. NanoDrop (Thermo Fisher Scientific, model: ND-1000 spectrophotometer)
  48. Power Pac and electrophoresis tank (Bio-Rad Laboratories)
  49. Tray
  50. Glass plate
  51. Tissue paper
  52. CL-1000, ultraviolet crosslinker (UVP)
  53. Hybaid Maxi 14 Hybridization Oven (Thermo Fisher Scientific)
  54. Hybridization tubes
  55. Kodak Biomax cassette (Eastman Kodak Company)
  56. 柯达X-OMAT 2000处理器(Eastman Kodak公司)

程序

  1. RNA提取
    1. 将细胞接种在直径为100mm的培养皿中,并在37℃下用2PFU的活IBV /细胞或相同量的UV灭活的IBV(UV-IBV)感染。 通过在感染后1小时更换新鲜培养基除去培养基中的过量病毒。
    2. 将IBV感染的细胞在37℃下在5%CO 2中孵育。
    3. 在指定的时间点(感染后0,2,4,8,12,16,20,24,28h),将细胞用10ml磷酸缓冲盐水(PBS)缓冲液漂洗一次 并在室温下在1ml TRIzol中裂解5分钟 温度。
    4. 将细胞裂解物转移到eppendorf管中,并向每个管中加入五分之一(体积/体积)的氯仿。
    5. 用手剧烈摇动管15秒,并在室温下温育3分钟,然后在4℃以12,000×g离心15分钟。
    6. 将上层水相转移到新管中并与1:1(体积/体积)的100%异丙醇混合,然后在室温下温育10分钟。
    7. 通过在4℃以12,000×g离心10分钟沉淀RNA。
    8. 用1ml 70%无RNA酶的乙醇洗涤RNA沉淀一次,并以7500×g离心5分钟。
    9. 将RNA沉淀物空气干燥并通过在65℃下孵育15分钟溶于100μl无RNA酶的H 2 O中。
    10. RNA浓度和纯度通过NanoDrop测定。
    11. 将RNA储存在-80℃用于进一步使用。

  2. 探针制备
    1. 通过RT-PCR获得Northern印迹探针,并使用DIG标记试剂盒通过地高辛(DIG)标记,如下所述。
    2. 在最终体积为10.5μl的无菌0.2ml薄壁Gene-Amp PCR管中,将2μg总RNA加入2μl10pmol寡核苷酸(dT)中。
    3. 在65℃变性10分钟后,立即在冰上冷却试管。
    4. 然后将变性的RNA-引物混合物加入到最终体积为20μl的反应混合物中,该反应混合物含有10mM dNTP,20单位的Rnasinα核糖核酸酶抑制剂,1x Expand反转录酶缓冲液和50单位逆转录酶。
    5. 第一链cDNA在43℃合成1小时,反应可以通过在65℃加热10分钟终止(任选)。
    6. 使用DIG标记试剂盒,根据制造商的手册,通过聚合酶链反应(PCR)在25或50μl含有合适的引物对和PFU聚合酶的反应中实现cDNA的扩增。
    7. 用于人ATF4的引物是5'-CCGTCCCAAACCTTACGATC-3'(正向)和5'-ACTATCCTCAACTAGGGGAC-3'(反向)。用于人ATF3的引物是5'-GGTTAGGACTCTCCACTCAA-3(正向)和5'-AGACAGTAGCCAGCGTCCTT-3'(反向)。用于人GADD153的引物是5'-GATTCCAGTCAGAGCTCCCT3'(正向)和5'-GTAGTGTGGCCCAAGTGGGG-3'(反向)。准备每个相应PCR引物的10倍浓度溶液
    8. 将以下试剂按以下顺序在冰上的0.2ml反应管中加入:ddH 2 O32.25μl,PCR缓冲液5μl,PCR DIG标记混合物5μl,正向引物5μl,反向引物5μl,酶混合物0.75μl,模板cDNA2μl,终体积50μl。涡旋混合物并短暂离心
    9. 将样品置于热块循环仪中并在以下条件下进行PCR:在95℃初始变性2分钟,在95℃变性10秒,在60℃退火30秒,并在72℃延伸2分钟,重复变性,退火和延伸30个循环,最后在72℃延伸7分钟。
    10. 在琼脂糖微量凝胶上运行每个PCR反应的一部分,然后用溴化乙锭染色凝胶,并在UV下检查PCR产物。

  3. Northern印迹
    1. 为了通过Northern印迹分析RNA表达,将来自每个样品制备物的30μgRNA与RNA加载缓冲液混合,并加载在1.3%琼脂糖甲醛凝胶(参见Recipes)中的孔上。
    2. 运行凝胶用3-4 V/cm在无RNA酶的凝胶盒中4小时,直到RNA被充分分离。
    3. 在0.25 - 0.5μg/ml溴化乙锭中简单染色凝胶,并在紫外光下检查凝胶
    4. 在20x SSC中漂洗凝胶2×15分钟,并通过用20×SSC在室温下过夜通过毛细管转移将凝胶上的RNA转移到Hybond TM超-N +膜上。
    5. 通过紫外线交联将RNA固定在膜上。 所使用的能量在245nm下为20,000μJoules/cm 2
    6. 在UV交联之后,在ddH 2 O中短暂冲洗膜并允许空气干燥。
    7. 预杂交膜用DIG easy Hyb 30分钟,轻轻搅拌68℃。
    8. 通过煮沸5分钟并在冰中快速冷却来变性DIG标记的RNA探针,并将变性探针(25ng/ml)加入到10ml预热的DIG Easy Hyb中。
    9. 将10ml探针/杂交混合物加入膜中,并在轻轻搅拌下在68℃温育6小时。
    10. 杂交后,在25℃下在恒定搅拌下用2×SSC,0.1%SDS洗涤膜2×5分钟,然后在68℃下在恒定搅拌下用0.1×SSC,0.1%SDS洗涤2×15分钟。
    11. 然后将膜在洗涤缓冲液中短暂漂洗(5分钟),并在封闭缓冲液中封闭30分钟。
    12. 封闭后,将膜与DIG抗体(稀释的抗DIG-AP1:10,000在封闭缓冲液中)温育30分钟,在洗涤缓冲液中洗涤2×15分钟,并在检测缓冲液中平衡3分钟。
    13. 根据制造商的说明书,使用CDP-Star检测信号(参见图1-3)。


      图1 。 在IBV感染的细胞中ATF4 mRNA的Northern印迹分析.Vero和H1299细胞用IBV(MOI〜1)感染,并在指定的时间点收获。分离总RNA并使用ATF4探针进行Northern印迹。将28S rRNA和18S rRNA的溴化乙锭染色显示为加样对照。确定ATF4的条带强度并归一化为rRNA

      图2. 在IBV感染的细胞中ATF3 mRNA的Northern印迹分析。如图1所示感染Vero和H1299细胞。如图1所示进行RNA提取和Northern印迹1使用ATF3探针。


      图3. 在IBV感染的细胞中,mRNA水平的GADD153的Northern印迹分析。 用IBV(MOI〜1)感染Vero和H1299细胞或用UV-IBV孵育并在指定的时间点收获。 RNA提取和Northern印迹如图1使用GADD153探针进行

食谱

  1. 10×TAE电泳缓冲液(1L)
    48.4g三(羟甲基)氨基甲烷(Tris碱) 11.4ml 17.4M冰醋酸
    3.7g EDTA,二钠盐
    ddH sub 2 O
  2. 10x MOPS缓冲液(1 L)
    83.7g 3-(N-吗啉代)丙磺酸(MOPS)
    13.61g乙酸钠。3 H 2 O 2 3.7 g EDTA
    ddH sub 2 O
  3. 1x MOPS缓冲区(1 L)
    100 ml 10x MOPS缓冲液
    20ml 37%甲醛
    880ml ddH 2 O 2·h/v
  4. 1.3%甲醛琼脂糖凝胶 1.3克琼脂糖 10ml 10×甲醛琼脂糖凝胶缓冲液
    将无RNase的水加至100 ml
    加热混合物以熔化琼脂糖
    在水浴中冷却至65℃。
    加入1.8ml的37%(12.3M)甲醛(1当量)和1μl的10mg/ml溴化乙锭储液
    充分混合并倒入凝胶支架上
    在运行凝胶之前,在1x甲醛琼脂糖凝胶运行缓冲液中平衡至少30分钟
  5. 20x SSC缓冲液(1L)
    175.3g NaCl
    88.2g柠檬酸钠
    ddH sub 2 O
    用几滴14N HCl溶液调节pH至7.0 高压灭菌
    灭菌
  6. 2×SSC,0.1%SDS(1L) 100ml 20x SSC缓冲液
    10ml 10%SDS
    890ml ddH 2 O·dm/h
  7. 0.1x SSC,0.1%SDS 10ml 20×SSC缓冲液
    10ml 10%SDS
    980ml ddH 2 O 2 /

致谢

该协议改编自先前的出版物Liao等人(2013)。 这项工作得到国家研究基金会的竞争研究计划(CRP)资助(R-154-000-529-281)的部分支持, 新加坡。

参考文献

  1. Liao,Y.,Fung,T. S.,Huang,M.,Fang,S. G.,Zhong,Y.和Liu,D. X.(2013)。 在冠状病毒感染性支气管炎病毒感染期间上调CHOP/GADD153通过限制细胞外信号传导的细胞活化来调节细胞凋亡, 调节的激酶通路。 J Virol 87(14):8124-8134。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liao, Y., Fung, T. S., Huang, M., Fang, S., Zhong, Y. and Liu, D. (2014). RNA Isolation and Northern Blot Analysis. Bio-protocol 4(6): e1077. DOI: 10.21769/BioProtoc.1077.
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