JP2012511145A - ホルマリン固定組織からの種々の生体分子の並行抽出 - Google Patents
ホルマリン固定組織からの種々の生体分子の並行抽出 Download PDFInfo
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Abstract
Description
a)出発材料の前記架橋を分解する工程、
b)出発材料に存在する異なる生体分子を少なくとも1つの画分(A)および少なくとも1つの画分(B)に分離する工程および
c)工程b)の前記画分(A)および(B)の少なくとも1つから異なる生体分子を単離または検出する、あるいは、単離および検出する工程、を含む方法、
および前記方法を実施するためのキットによって達成される。好ましい実施形態は従属クレームに含まれる。
本発明の方法による生体分子の分離
本実験は、ラット肝臓由来のホルマリン固定パラフィン包埋組織サンプル(FFPEサンプル)を使用した。前記サンプルから厚さ約10μmの厚さの切片をミクロトームを用いて調製し、各サンプルから2つの切片を使用した。その後前記FFPE切片からのタンパク質およびDNA/RNAの単離では、QIAGEN製のRNeasy FFPEキット、QIAamp FFPEキット、Qproteome FFPEキットおよびAllprep DNA/RNA Miniキットの各成分を使用した。
本発明の方法により分画された生体分子のゲル解析。
本発明の方法により分画されたRNAのリアルタイムRT−PCR分析
核酸の単離だけでなく増幅による分析に対する本発明の方法の効果を調査するため、実施例1に記載の方法によりペレット画分および上清画分から単離されたRNAを使用してさらにリアルタイム定量RT−PCRによる分析を行った。
Claims (14)
- 架橋により固定された同じ生物学的出発材料から様々な種類の生体分子を並行精製する方法であって、
a)前記出発材料の前記架橋を分解する工程、
b)前記出発材料に存在する前記異なる生体分子を少なくとも1つの画分(A)および少なくとも1つの画分(B)に分離する工程および
c)工程b)の前記画分(A)および(B)の少なくとも1つから異なる生体分子を単離または検出/分析する、あるいは、単離および検出/分析する工程、
が行われる、方法。 - 前記出発材料の前記架橋は以下の工程:
(i)前記材料を好ましくは水性の溶液に移す工程、
(ii)前記材料を、前記溶液中、前記可溶性タンパク質を遊離するのに十分な温度でインキュベートする工程
(iii)その後さらに60℃を超える温度でインキュベートする工程
により、工程a)で分解される、請求項1に記載の方法。 - 工程(i)で使用される前記溶液は界面活性剤を含み、タンパク質分解活性を有する化合物を含まないバッファーである、請求項2に記載の方法。
- 工程(ii)における前記材料は前記溶液中で煮沸される、請求項2または3に記載の方法。
- 工程b)において可溶性画分(A)および不溶性画分(B)への分離が行われる、請求項1〜4のいずれかに記載の方法。
- 画分(A)と画分(B)との分離により、少なくとも前記出発材料から遊離可能な前記タンパク質が、少なくとも遊離可能な前記核酸から実質的に定量的に分離され、前記可溶性タンパク質は実質的に定量的に画分(A)中にある一方、前記可溶性核酸は実質的に定量的に画分(B)中にある、請求項1〜5のいずれかに記載の方法。
- 工程c)による前記異なる生体分子の単離はタンパク質精製またはタンパク質単離の慣用される方法を用いて画分(A)からのタンパク質の精製/単離により行われ、前記不溶解性サンプル成分を破壊する少なくとも1つの追加処理、および/またはその後前記出発材料中になお残存している架橋を分解すること、さらに前記可溶性核酸を単離することを含むその後の工程d)により、画分(B)から核酸を単離する、請求項1〜6のいずれかに記載の方法。
- 前記タンパク質の精製/単離はクロマトグラフィ法、電気泳動による分離、タンパク質結合材料への特異的結合、特異抗体を用いたタンパク質の捕捉または沈殿により行われ、前記核酸の単離は沈殿、核酸結合材料への結合、電気泳動またはクロマトグラフィにより実施される、請求項1〜7のいずれかに記載の方法。
- タンパク質の検出/分析は免疫学的方法、クロマトグラフィ法、シーケンシングまたは電気泳動によって行われ、前記核酸の検出/分析は特異的PCR、電気泳動、ハイブリダイゼーション法またはシーケンシングによって行われる、請求項1〜8のいずれかに記載の方法。
- 請求項1〜9のいずれかに記載の方法を実施するためのキットであって、少なくとも(1)前記出発材料の前記架橋を分解するための、プロテアーゼを含まない水性系、好ましくはバッファー系、(2)場合により前記溶液(1)に存在していてもよい界面活性剤、および(3)プロテアーゼを含む溶液またはプロテアーゼを含む、キット。
- (4)少なくとも1種の核酸結合材料、(5)核酸を単離するための追加のバッファー、好ましくは結合バッファーおよび溶出バッファーをさらに含む、請求項10に記載のキット。
- 前記水性系(1)は少なくとも1種の還元試薬、少なくとも1種の界面活性剤、および少なくとも1種の求核試薬を含むバッファーである、請求項10または11に記載のキット。
- 生物学的サンプルに含まれる生体分子を分析および/または定量するための、請求項10〜12のいずれかに記載のキットの使用。
- ヒトの体外のサンプルまたは動物の体外のサンプルに基づき疾患の診断、疾患の予後診断、疾患に対する治療の決定、および疾患に対する治療のモニタリングを行うための、請求項10〜12のいずれかに記載のキットの使用。
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CN102238991B (zh) | 2015-11-25 |
EP2373390B1 (de) | 2016-10-05 |
US10011826B2 (en) | 2018-07-03 |
EP2373390A1 (de) | 2011-10-12 |
EP2193831A1 (de) | 2010-06-09 |
WO2010063837A1 (de) | 2010-06-10 |
JP5763545B2 (ja) | 2015-08-12 |
JP2014209919A (ja) | 2014-11-13 |
US20110244468A1 (en) | 2011-10-06 |
CN102238991A (zh) | 2011-11-09 |
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