JP6324984B2 - パラフィン包埋サンプルを処理する方法 - Google Patents
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Description
本発明の態様に基づくプロセスを用いたFFPEサンプルに由来する核酸(この場合DNA)の収率を、Promega ReliaPrep FFPE gDNA Miniprepシステムを用いて得られた収率と比較する比較試験を実施した。
本発明の態様に基づくプロセスを用いて、FFPE腎臓組織サンプルから得られた10ミクロンの切片セクションに由来するRNAの収率を、従来型のPROMEGA ReliaPrep FFPE全RNA Miniprepシステムを用いて得られた収率と比較するために、実施例1に記載した試験と類似した試験を実施した。
Covaris FFPE DNA精製キットを用いて、FFPEサンプルからDNAを精製及び回収し、定量PCR(qPCR)を実施するために、これをマルチプレックスPCR準拠品質管理アッセイ法により試験した。溶媒に基づくパラフィン除去法を採用するQIAGEN QIAamp DNA FFPE組織キットを用いて得られた収率の2〜4倍上回る収率で、精製DNAを回収した。
別の実施例では、集束型音響によるFFPE組織からのパラフィン分離を試験するのに蛍光顕微鏡検査を用いた。正常なヒト腎臓から得られたFFPE組織サンプルは、Cooperative Human Tissue Network、Eastern Division University of Pennsylvaniaより提供された。厚さ約25ミクロンのFFPEサンプルを切り出すのにミクロトームを用いた。
Claims (14)
- 予めホルマリン固定されパラフィンに包埋されたパラフィン包埋組織サンプルを、容器内に供給する工程と、
前記パラフィンを溶解しない前記パラフィンに対して非溶剤の溶液を、前記パラフィン包埋組織サンプルを伴った前記容器内に供給する工程と、
前記容器内のサンプル及び非溶剤の溶液を、前記サンプルからパラフィンを分離するための約100キロヘルツ〜約100メガヘルツの周波数及び集束帯を有する音響エネルギーに曝露することにより、前記パラフィン包埋組織サンプルからパラフィンを分離する工程と、
前記サンプルからパラフィンを分離した後の前記サンプルから生体分子を回収する工程と、
を含む、パラフィン包埋サンプルを処理する方法。 - 前記分離する工程が、核酸及び/又はプロテオミック物質が前記サンプルから回収できるように、十分量のパラフィンを前記サンプルから分離するのに十分な時間にわたって前記サンプルを集束型音響エネルギーに曝露する工程を含む、請求項1に記載の方法。
- 前記分離する工程が、前記サンプルに付着したパラフィンの90%超を分離する工程を含む、請求項1に記載の方法。
- 前記分離する工程が、前記サンプルを集束型音響エネルギーに曝露している間、前記サンプルを再水和する工程を含む、請求項1に記載の方法。
- 前記分離する工程が、前記容器が約5〜60℃の温度の液体の浴槽内に置かれている間、前記サンプルを集束型音響エネルギーに曝露する工程を含む、請求項1に記載の方法。
- 前記分離する工程が、前記サンプルの温度を約60℃未満に維持する工程を含む、請求項1に記載の方法。
- 前記サンプルからパラフィンを分離した後、プロテアーゼを前記容器内の非溶剤の溶液及びサンプルに添加する工程をさらに含む、請求項1に記載の方法。
- プロテアーゼ含有溶液を前記サンプルと混合するために、前記サンプル及び前記プロテアーゼ含有溶液を集束型音響エネルギーに曝露する工程をさらに含む、請求項7に記載の方法。
- 前記サンプル及びプロテアーゼ含有溶液を集束型音響エネルギーに曝露する工程が、前記サンプルに由来する核酸物質をより小さな断片に断片化するのに適する集束型音響エネルギーに、前記サンプルを曝露する工程を含む、請求項8に記載の方法。
- 前記容器が、回収工程期間中に、前記サンプルから分離したパラフィンを含有する、請求項9に記載の方法。
- 前記回収工程が、前記サンプル、分離したパラフィン並びに核酸及び/又はプロテオーム物質の非溶剤の溶液を前記容器からピペッティングする工程を含む、請求項9に記載の方法。
- 前記容器内のサンプルを、プロテアーゼ含有溶液と共に、前記サンプル中のホルムアルデヒド架橋を元に戻す温度でインキュベートする工程をさらに含む、請求項7に記載の方法。
- 前記サンプル中の組織を消化するために、前記容器内のサンプルをプロテアーゼ含有溶液と共に、50〜60℃の温度でインキュベートする工程をさらに含む、請求項7に記載の方法。
- 前記集束帯が、約2センチメートル未満の幅を有し、前記音響エネルギーが、前記容器とは間隔が置かれ且つ前記容器の外部にある音響エネルギー源に由来し、少なくとも前記音響エネルギーの一部分が、前記容器外部に伝播する、請求項1に記載の方法。
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US13/678,755 US9080167B2 (en) | 2012-11-16 | 2012-11-16 | System and method for processing paraffin embedded samples |
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PCT/US2013/070290 WO2014078650A1 (en) | 2012-11-16 | 2013-11-15 | System and method for processing paraffin embedded samples |
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US10370635B2 (en) | 2012-03-15 | 2019-08-06 | Flodesign Sonics, Inc. | Acoustic separation of T cells |
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WO2014078650A1 (en) | 2014-05-22 |
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