浅谈northern blot-2

1、Northern Blot for microRNA 营养与食品卫生学教研室 路慧敏 Biogenesis and Action of miRNAs Conventional Detecting Techniques Northern Blot Quantitative Real time-RT-PCR History Northern blot Eastern blot Southern blot Western blot ? Northern Blot Strategy Harvest tissues/cells and extract RNA Electrophoresis and bl

2、otting Design SDS 0.1% at 15-25C. 2. Wash 2 15 min in 0.5 SSC; SDS 0.1% at50C under constant agitation. 3. After hybridization and stringency washes, rinse membrane briefly 5 min in Washing buffer, at room temperature. 4. Incubate for 30 min in Blocking solution, at room temperature. 5. Incubate for

3、 30 min in Antibody solution (1:10000), at room temperature 6. Wash 2 15 min in Washing buffer, at room temperature. 7. Equilibrate 5 min in Detection buffer. 8. Place membrane with RNA side facing up on a development folder (or hybridization bag) and apply diluted CSPD solution(1:100). 9. Squeeze o

4、ut excess liquid and seal the edges of the development folder. 10. Incubate the damp membrane for 5-15 min at 37C to enhance the luminescent reaction. 11.Expose to imaging instrument or to X-ray film for e.g., 525 min at 1525C. Post hybridization washes and detection Northern Blot Strategy Harvest t

5、issues/cells and extract RNA Electrophoresis and blotting Design & label probe of known sequence Hybridization Visualize & quantify signal Stripping and ReprobingStripping and Reprobing the Membrane the Membrane l Incubate4,5 in 50% formamide (deionized)/5% SDS/50 mM Tris-HCl (pH 7.5), 2 x 60 min, 8

6、0C, in a sealed bag. l Rinse in 2x SSC, = 5 min, RT. Store in 2x SSC. l An alternative stripping solution for RNA probes is 90% formamide/10 mM sodium phosphate (pH 7.5). 毒毒 0.溴化乙锭（EB）：大家都比较熟悉了。具有强诱变致癌性，使用时一定要 戴一次性手套，注意操作规范，不要随便触摸别的物品。 1.DEPC(焦碳酸二乙酯):闻起来香香甜甜的,可是害人不眨眼!一种强有力的 蛋白质变性剂,而且怀疑是致癌剂.开瓶时将瓶子远离你

7、,内压可导致溅泼.操作 时戴合适的手套,穿工作服,并在化学通风橱里进行. 2.甲醛甲醛：毒性较大且易挥发，也是一种致癌剂，易通过皮肤吸收，对眼睛、 粘膜和上呼吸道有刺激和损伤作用。避免一如其挥发的气雾。戴手套和护目 镜。始终在通风橱中操作。远离热、火花及明火。 3.TRIzol ，对眼睛有刺激性，腐蚀皮肤。有一次不小心溅出一小滴在脸上， 马上就红了，过一会儿感觉到疼，一周之后才好。如果溅上，马上用大量的 水冲洗。 4.双丙烯酰胺双丙烯酰胺，神经毒剂，效应累积 5.过硫酸酸铵过硫酸酸铵：对粘膜和上呼吸道、眼睛和皮肤又较大危害性，吸入可致命。 操作时戴手套、护目镜。始终在通风橱中操作。 6.TEM

8、ED 7.beta-巯基乙醇巯基乙醇，可致命，对呼吸道、皮肤和眼睛有伤害 大家在实验室里，接触到的化学试剂多半都有危害性，紫外灯，如果离心机 不是很好的话的噪声污染，VORTEX会导致手指需要震动，等等，所以每个 人都应该注意保护自己，而不能因为图省事或者别的什么原因而放松对安全 的注意。另外就是注意规范操作。 / / http:/ / Troubleshooting Inefficient probe labeling Concentration

9、 of labeled probe too low Inefficient hybridization Insufficient exposure time Low sensitivity Troubleshooting Inefficient labeling Purify template by phenol/chloroform extraction. No EDTA in the buffers used for storage of template. probe not contain crosshybridizing vector sequences. Concentration

10、 of labeled probe too high 100 ng labeled RNA probe or25 ng labeled DNA probe/ml hybridization buffer). Determine optimal probe concentration experimentally. Target concentration in sample too high Ineffective stringency washes Always prewarm wash solution to correct temperature. Check temperature o

11、f stringency washes. Membrane dried during procedure High background Troubleshooting Spotty background Antibody contained high molecular weight “complexes” rochedig120-136.pdf Always centrifuge antibody stock solution for 5 min at full speed before taking aliquot (of supernatant) to make Antibody So

12、lution. SSC crystals Wash membrane briefly in 2x SSC before fixation by baking. Protein contamination in probe Filter labeled probe through a 0.45 m cellulose acetate filter (e.g., from Schleicher and Schuell). Purify probe with the High Pure PCR Product Purification Kit. Electrophoresis and blottin

13、g Agarose Gel *10XMOPS, pH7.0 (adjusted with NaOH) 200mM MOPS buffer 50mM sodium acetate 20nM EDTA loading buffer(2X) 250ul Deionized formamide 83ul 37% formaldehyd 50ul 10 X MOPS 50ul 100% Rnase-free Glycerol 10ul 2.5% Bromophenol blue 57ul DEPC-treated water 20XSSC buffer, pH7.0 3M NaCl 300mM sodi

14、um citrate Detection of Hybridization Probes on Detection of Hybridization Probes on a Blota Blot 2 2SSCSSC、0.10.1SDSSDS，室温洗涤，室温洗涤2 25min5min 0.10.1SSCSSC、0.10.1SDSSDS，6868C C洗涤洗涤2 215min15min Washing BufferWashing Buffer，室温快速洗涤，室温快速洗涤1 1次次 Blocking SolutionBlocking Solution，室温孵育，室温孵育30min30min Washing BufferWashing Buffer，室温洗涤，室温洗涤2 215min15min 在在Detection BufferDetection Buffer中平衡中平衡2 25min5min 将膜放入封口膜中，立即均匀滴加约将膜放入封口膜中，立即均匀滴加约0.6ml0.6ml的的CDP-StarCDP-Star，封口，封口 将膜放入暗盒中，压片约将膜放入暗盒中，压片约1min1min，冲片，读片，冲片，读片 The northern blot technique was develope