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In situ hybridization is an effective method to examine the expression level and location of gene of interest on tissues or cells. The method provided in this protocol is a detailed description of synthesizing antisense probe for in situ hybridization.

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[Bio101] Dig RNA Probe Synthesis and Purification
[Bio101] 地高辛 RNA探针的合成和纯化

分子生物学 > RNA > RNA 合成
作者: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
1/20/2012, 14777 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.173

[Abstract] In situ hybridization is an effective method to examine the expression level and location of gene of interest on tissues or cells. The method provided in this protocol is a detailed description of synthesizing antisense probe for in situ hybridization.

[Abstract] 原位杂交技术是检查组织或细胞中基因表达水平和位置的一种有效的方法。在这个程序中提供的方法详细描述了一种合成反义探针的原位杂交。

Materials and Reagents

  1. CI (Chloroform: Isoamylalcohol = 24:1)
  2. NaOAc
  3. EtOH
  4. LiCl
  5. DEPC H2O
  6. RNasin Plus Protease Inhibitor (Promega Corporation, catalog number: N2611 )
  7. 70% ethanol
  8. PCI (Phenol: Chloroform: Isoamylalcohol = 25:24:1, volume, Sigma-Aldrich, catalog number: P3802
  9. Glycogen (QIAGEN, catalog number: 158930 )
  10. DIG RNA labeling kit (Roche Diagnostics, catalog number: 11175025910 )
  11. Quick Spin Columns (Roche Diagnostics, catalog number: 1274015 )

Equipment

  1. Standard tabletop centrifuges
  2. RNase-free Eppendorf tube
  3. NanoDrop
  4. Falcon snap cap tube
  5. Water bath

Procedure

  1. Linearizing the cDNA
    1. Cut 5 μg of cDNA in a 100 μl reaction. I use ~2-3 μl of enzyme.
    2. Add 1 volume (100 μl) PCI, vortex and centrifuge 2 min.
    3. Transfer upper phase in a new tube and add 1 volume (100 μl) CI, vortex and centrifuge 1 min.
    4. Transfer upper phase to new tube. Ethanol precipitate this phase.
    5. Add 200 μl EtOH, 10 μl 3 M NaOAc, 0.5 μl 1 mg/ml glycogen and precipitate at -20 °C overnight.
    6. Spin down and wash with 70% ethanol.
    7. Spin again and resuspend in 11 μl nuclease-free water. Take 1 μl of linearized DNA to spec the concentration.

  2. DIG probe synthesis (using the Roche labeling kit)
    * = provided with kit
    1. Add the following to an RNase-free Eppendorf tube (20 μl reaction):
      1 μg linearized DNA template
      2 μl NTP labeling mix*
      2 μl transcription buffer*
      2 μl of RNA polymerase (T7, T3, SP6)*
      1 μl RNase inhibitor*
      RNase-free water to 20 μl total volume.
    2. Mix by flicking tube gently and briefly centrifuge.
    3. Incubate reaction at 37 °C for 2 h.
    4. Add 2 μl DNase I* to the reaction.
    5. Incubate for 15-20 min at 37 °C.

  3. Purifying the DIG probe
    Method 1: Precipitation of RNA probe
    1. Add:
      3.7 μl DEPC H2O
      1.33 μl 7.5 M LiCl
      75 μl EtOH
    2. Precipitate at -20 °C O/N.
    3. Centrifuge 4 °C, 13,000 rpm for 30 min.
    4. Wash pellet in 500 μl 70% DEPC-EtOH (-20 °C).
    5. Centrifuge 5 min, 13000 rpm, RT.
    6. Air dry 5-10 min.
    7. Add:
      50 μl DEPC H2O
      20 units RNasin (0.35 μl)
      Resuspend RNA probe
      Take 2 μl to check on agrose gel and quantify RNA using NanoDrop.
    8. Store RNA probe at -20 °C.
      Method 2: Quick Spin Columns

    1. Remove the quick spin columns from the storage bag and gently invert several times to resuspend the medium.
    2. Remove the top cap from the column and then the bottom cap. Some of the buffer will run out of the column. Discard it.
    3. Place the column in one of the collection tubes (provided in kit) and put both the column and collection tube into a falcon snap cap tube (clear tube, like the ones we use for maxi preps).
    4. Spin the sample in the table-top centrifuge at 1,100 x g for 2 min.
    5. Discard the buffer and if the tip of the column is submerged in the buffer when you take it out of the first spin, then spin it again to make sure that all of the buffer is cleared from the column.
    6. Place the column in a new collection tube (provided with kit).
    7. Add your DIG probe to the center of the column without touching the beads.
    8. Spin for an additional 2 min at 1,100 x g. The eluate from this spin is your DIG probe sample. Transfer it to a clean Eppendorf tube (about 20 μl) and bring up to 100 μl with nuclease-free water. This will give you a concentration of about 100 ng/μl of probe.

Notes

I usually synthesize 3-4 separate dig probe reactions of the same probe and add all of them to the same G50 column. This way you have made quite a bit of probe using only 1 column.

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.

材料与试剂.

 

1.        PCl:氯仿:异戊醇= 25:24:1,体积,Sigma公司P3802

2.        CI(氯仿:异戊醇= 24:1

3.        乙醇

4.        NaOAc

5.        糖原(Qiagen公司158930

6.        RNA标记试剂盒(Roche 11175025910

7.        快速离心柱(Roche货号#1 274 015

 

设备

 

1.        RNaseEppendorf

2.        快速离心柱(Roche货号#1 274 015

3.        离心机

4.        水浴

 

步骤

 

1.        线性化的cDNA

1)      剪切5微克的cDNA100μl反应,用?2-3μl的酶。

2)      添加1卷(100微升)PCI,涡旋离心2分钟

3)      在一个新的管上部转移,并添加1卷(100微升)Cl,旋涡和离心1分钟

4)      转让上相至新管.

5)      加入200μl乙醇,0.5μl 1 mg / ml的糖原和沉淀,在10μl  3 M NaOAc - 20°C过夜。

6)      旋转,并用70%乙醇清洗。

7)      旋转和重悬在11μl无核酸酶水。取1μl线性化DNA观察浓度。

2.        地高辛探针合成:(使用Roche标记试剂盒)

* =提供的试剂盒

1)      添加下面的一个无RNaseEppendorf管:(20μl反应体系)

1微克的线性DNA模板

2μl NTP标签组合*

2μl转录缓冲液*

2μl RNA聚合酶(T7T3SP6*

1μl RNase抑制剂*

RNase水添至20μl总体积

2)      轻轻弹混合管,并短暂离心

3)      37孵育反应2小时。

4)      加入2μl DNA I*到反应体系。

5)      37°C孵育15-20分钟

3.        净化地高辛探头:

方法1:沉淀RNA探针

1)      加入:

a.      3.7μl DEPC处理水

b.      1.33微升7.5M氯化锂

c.      75μl乙醇

2)      沉淀在-20 °C  O / N

3)      离心4°C13000 RPM 30分钟

4)      500μl 70DEPC -乙醇(-20°C)清洗沉淀

5)      离心5 min13000 RPMRT

6)      空气干燥5-10分钟

7)      加入:

a.      50μl  DEPC处理水

b.      20个单位RNasin0.35微升)

c.      重悬的RNA探针

8)      2μl检查琼脂糖凝胶和使用nanodrop量化RNA

9)      储存RNA探针在-20°C

方法2(快速离心柱,Roche1 274 015

1)      快速自旋柱从储存袋中取出,轻轻颠倒几次,悬浮介质。

2)      拆下柱的顶盖和底盖。有些柱的缓冲液已经耗尽则丢弃。

3)      将柱放在收集管(套件)中,并把柱和收集管放进管中(清洗管)。

4)      在桌面的离心机上,1100 X g旋转样品2分钟(这是现在旁边的地板胶机真空泵)

5)      丢弃的缓冲液,如果柱的一角是淹没在缓冲液中,当你第一次旋转它,然后再旋转,以确保所有柱中的缓冲液都被清除。

6)      将柱放置在一个新的收集管(试剂盒提供)。

7)      添加您的地高辛探针至中心柱而不触及的珠子。

8)      1100 X g 另外旋转2分钟。这个旋转的洗脱液是您的挖掘探测样本。转移到一个干净的Eppendorf管(约20μl),并带来高达100μl无核酸酶水。这会给你一个约100 ng/μl的探针浓度。

新增注:我通常同一个探针合成3-4单独地高辛反应探针,并都添加到G50柱。用这个方法你只要用1柱就行了。

 

 

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How to cite this protocol: Jing, L. (2012). Dig RNA Probe Synthesis and Purification. Bio-protocol Bio101: e173. DOI: 10.21769/BioProtoc.173; Full Text



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