RNA Isolation and Northern Blot Analysis
RNA分离和Northern印迹杂交分析
发布: 2014年03月20日第4卷第6期 DOI: 10.21769/BioProtoc.1077 浏览次数: 32470
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jul 2013
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
- Vero cells (kidney epithelial cells extracted from an African green monkey) (ATCC, catalog number: CCL-81 TM)
- H1299 cells (human lung carcinoma cell line) (ATCC, catalog number: CRL-5803 TM)
- The egg-adapted Beaudette strain of IBV (ATCC, catalog number: VR-22 )
- 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. - 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. - Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 25200-072 )
- TRIzol reagent (Life Technologies, Gibco®, catalog number: 15596-018 )
- Chloroform (Thermo Fisher Scientific, catalog number: C/4960/17 )
- Isopropanol (Thermo Fisher Scientific, catalog number: P/7507/17 )
- Ethanol (Merck KGaA, catalog number: 1.00983.2500 )
- RNase free water
- Reverse transcriptase AMV (Roche Diagnostics, catalog number: 10109118001 )
- Oligo (dT) (1st Base Biochemicals)
- RNasin® ribonuclease inhibitor (Promega Corporation, catalog number: N2511 )
- Primers (1st Base Biochemicals)
- DIG labeling kit (Roche, catalog number: 11175025910 )
- RNA loading buffer (New England Biolabs, catalog number: B0363S )
- Agarose (1st Base Biochemicals, catalog number: BIO-100-500G )
- Formaldehyde (Thermo Fisher Scientific, catalog number: F75P1GAL ))
- Ethidium bromide (Bio-Rad Laboratories, catalog number: 1610433 )
- HybondTM-N+ membrane (Amersham Biosciences, catalog number: RPN303B )
- DIG Wash and Block Buffer Set (Roche Diagnostics, catalog number: 11585762001 )
- DIG easy Hyb (Roche Diagnostics, catalog number: 11603558001 )
- Anti-digoxigenin-AP fab fragments (Roche Diagnostics, catalog number: 11093274910 )
- CDP-Star (Roche Diagnostics, catalog number: 12041677001 )
- Amersham hyperfilm ECL (Amersham Biosciences, catalog number: 28906837 )
- 70% RNase-free ethanol
- Tris(hydroxymethyl)aminomethane (Tris base) (Promega Corporation, catalog number: H5135 )
- Acetic acid (Glacial) (Merck KGaA, catalog number: 1.00063.2500 )
- 3-(4-morpholino) propane sulfonic acid (MOPS) (Thermo Fisher Scientific, catalog number: BP308-500 )
- Sodium acetate.3H2O (Thermo Fisher Scientific, catalog number: S207-10 )
- Sodium Citrate (Thermo Fisher Scientific, catalog number: S25545 )
- 10x TAE Electrophoresis Buffer (1 L) (see Recipes)
- 10x MOPS buffer (1 L) (see Recipes)
- 1x MOPS buffer (1 L) (see Recipes)
- 1.3% Formaldehyde Agarose gel (see Recipes)
- 20x SSC buffer (1 L) (see Recipes)
- 2x SSC, 0.1% SDS (1 L) (see Recipes)
- 0.1x SSC, 0.1% SDS (see Recipes)
Equipment
- 100 mm cell culture dishes (Corning, catalog number: 430167 )
- 0.2 ml thin-wall Gene-Amp PCR tube (Corning, Axygen®, catalog number: PCR-02-C )
- FormaTM Steri-CycleTM CO2 Incubators (Thermo Fisher Scientific, catalog number: 201370 )
- OLYMPUS CKX31 microscope
- Eppendorf centrifuge 5415R
- NanoDrop (Thermo Fisher Scientific, model: ND-1000 spectrophotometer )
- Power Pac and electrophoresis tank (Bio-Rad Laboratories)
- Tray
- Glass plate
- Tissue paper
- CL-1000, ultraviolet crosslinker (UVP)
- Hybaid Maxi 14 Hybridization Oven (Thermo Fisher Scientific)
- Hybridization tubes
- Kodak Biomax cassette (Eastman Kodak Company)
- Kodak X-OMAT 2000 processor (Eastman Kodak Company)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
微生物学 > 微生物遗传学 > RNA
分子生物学 > RNA > RNA 提取
分子生物学 > RNA > RNA 检测
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