Rapid quantitative PCR for determination of relative gene expressions in tissue specimens.
Rapid quantitative PCR for determination of relative gene expressions in tissue specimens.
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快速定量 PCR 测定组织样本中的相关基因表达。
DOI：
10.1101/gr.4.5.305
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Danenberg,PV
中科院分区：
文献类型：
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作者：
Lenz,HJ;Hill,C;Danenberg,KD;Leichman,LL;Priest,DG;Danenberg,PV
材料和方法 微型柱的尺寸和填充到柱中的 Sephadex 量经过设计，使得当 25 I~ 1 溶液加载到柱顶部时，离心后空隙体积中包含的大分子量物质将完全通过柱，但小分子则不会。为了准备离心微型柱，将已在 STE 缓冲液（0.1 M NaCl、10 mM Tris-HCl，pH 8.0、1 mM EDTA）中溶胀过夜的 Sephadex G-50 Fine（Sigma Chemical Co.，St. Louis，MO）填充至微型测试管（Sarstedt 72700）的顶部。使用过的移液器吸头架可以在填充微型柱时方便地固定它们。这样可以轻松地一次制备许多色谱柱。为了将Sephadex转移到柱中，使用缩短至刚好到达微管底部的移液器吸头，并从底部开始填充管。管子充满Sephadex后，用G23针（Becton-Dickinson，Rutherford，NJ）刺穿底部。当大部分缓冲液排出后，将管以 1000g 离心 5 分钟 (Beckman J6B)。（此时柱基质应看起来半干，但不应有任何裂纹或空隙。）然后为每个柱安装一个由 0.65 毫升和 1.7 毫升微量离心管组成的适配器，用热手术刀从管底部切出底部（Slick Seal 微量管，National Scientific，San Rafael, CA) 并放入液体闪烁瓶 (Fisher 033725) 中。将转录反应物(5μl)与20μl STE缓冲液混合。然后将25-〜1个样品施加到凝胶床表面的中心。（为了更容易观察柱负载，可以添加0.4％溴酚蓝。）然后将管在1000g（Beckman JB6）下离心10分钟，并将柱和适配器从闪烁瓶中取出。（来自转录反应的标记RNA与最初施加的体积相同（25μl）现在应该在闪烁中添加 275 lxl 双蒸水和 3 ml 液体闪烁混合物后（RIA-Solve II，RPI，4：305-3089，冷泉港实验室/出版社 ISSN 1054-9803/95 $5.00 PCR 方法和应用 305
MATERIALS AND METHODSThe sizes of the minicolumns and the amounts of Sephadex packed into the columns were designed so that when 25 I~ 1 of solution is loaded onto the top of the columns, large molecular-weight material that would be contained in the void volume would pass entirely through the column after centrifugation but small molecules would not. To prepare the centrifugal minicolumns, microtest tubes (Sarstedt 72700) were filled to the top with Sephadex G-50 Fine (Sigma Chemical Co., St. Louis, MO) that had been swelled overnight in STE buffer (0.1 M NaC1, 10 mM Tris-HC1 at pH 8.0, 1 mM EDTA). A used pipette tip rack makes a convenient holder for the minicolumns as they are being filled. This allows the easy preparation of many columns at a time. To transfer the Sephadex into the columns, pipette tips shortened to just reach the bottom of the microtubes were used and the tubes were filled starting from the bottom. After the tubes were filled with the Sephadex, the bottoms were punctured with a G23 needle (Becton-Dickinson, Rutherford, NJ). When most of the buffer had drained out, the tubes were centrifuged for 5 min at 1000g (Beckman J6B).(The column matrix should look semidry at this point but there should not be any cracks or air spaces.) Each column was then fitted with an adapter consisting of a 0.65-and a 1.7-ml microcentrifuge tube from which the bottoms had been cut out with a hot scalpel (Slick Seal microtubes, National Scientific, San Rafael, CA) and placed into a liquid scintillation vial (Fisher 033725). The transcription reaction (5 i~ l) was mixed with 20 ixl of STE buffer. The 25-~ 1 sample was then applied to the center of the gel bed's surface.(For easier observation of the column loading, 0.4% bromphenol blue may be added.) The tubes were then centrifuged for 10 min at lOOOg (Beckman JB6) and the column and adapter removed from the scintillation vial.(The labeled RNA from the transcription reaction in the same volume as originally applied (25 i~ l) should now be in the scintillation vial.) After addition of 275 lxl of double-distilled water and 3 ml of liquid scintillation cocktail (RIA-Solve II, RPI, 4: 305-3089 by Cold Spring Harbor Laboraton/Press ISSN 1054-9803/95 $5.00 PCR Methods and Applications 305