ES2539856T3 - Lisis mejorada y transcripción reversa para la cuantificación de mRNA - Google Patents
Lisis mejorada y transcripción reversa para la cuantificación de mRNA Download PDFInfo
- Publication number
- ES2539856T3 ES2539856T3 ES09802454.0T ES09802454T ES2539856T3 ES 2539856 T3 ES2539856 T3 ES 2539856T3 ES 09802454 T ES09802454 T ES 09802454T ES 2539856 T3 ES2539856 T3 ES 2539856T3
- Authority
- ES
- Spain
- Prior art keywords
- reverse transcription
- sample container
- target rna
- lysis
- mrna quantification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 238000010839 reverse transcription Methods 0.000 title abstract 3
- 230000009089 cytolysis Effects 0.000 title abstract 2
- 108020004999 messenger RNA Proteins 0.000 title 1
- 238000011002 quantification Methods 0.000 title 1
- 238000004113 cell culture Methods 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 239000002299 complementary DNA Substances 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 239000012139 lysis buffer Substances 0.000 abstract description 2
- 238000000034 method Methods 0.000 abstract description 2
- 230000001464 adherent effect Effects 0.000 abstract 2
- 230000003196 chaotropic effect Effects 0.000 abstract 2
- 239000003795 chemical substances by application Substances 0.000 abstract 2
- 238000003757 reverse transcription PCR Methods 0.000 abstract 1
- 238000003753 real-time PCR Methods 0.000 description 2
- 101150087690 ACTB gene Proteins 0.000 description 1
- 102100021699 Eukaryotic translation initiation factor 3 subunit B Human genes 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000673456 Homo sapiens 60S acidic ribosomal protein P0 Proteins 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- 101000896557 Homo sapiens Eukaryotic translation initiation factor 3 subunit B Proteins 0.000 description 1
- 101000988834 Homo sapiens Hypoxanthine-guanine phosphoribosyltransferase Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Método para realizar una RT-PCR para amplificar un RNA diana que comprende los pasos de a) el cultivo de una población de células adherentes en un recipiente de cultivo de células b) la lisis de dicha población de células adherentes que se supone que contiene dicho RNA diana en dicho recipiente de muestras con un tampón de lisis que comprende entre 0,05 M y 1 M de un agente caotrópico c) la adición de reactivos a dicho recipiente de muestras que son necesarios para realizar una reacción de transcripción inversa de forma que el agente caotrópico está presente en una concentración de aproximadamente de 10 a 60 mM en dicho recipiente de muestras, y la transcripción reversa de dicho RNA diana en la primera hebra de cDNA d) amplificar dicha primera hebra de cDNA mediante la realización de múltiples ciclos de un protocolo de termociclado.
Description
E09802454
18-06-2015
Las células HeLa humanas cultivadas fueron sembradas en una placa de cultivo celular de 96 pocillos con un número aproximado de 4000 células por vial y se dejaron crecer. Después de aproximadamente 48 horas, se retiró el medio de cultivo y se reemplazó después con RNA, y las células se almacenaron en la nevera durante 24 horas. El RNA se retiró posteriormente, y las células se lavaron con PBS frío. Se añadieron 12,5 l de tampón de lisis de
5 acuerdo con el ejemplo 1 y las células se incubaron durante 10 minutos a temperatura ambiente. Se añadieron reactivos de RT Transcriptor a un volumen final de RT de 50 l. En los controles sin RT controla la transcriptasa inversa se omitió de la mezcla RT. La placa de cultivo celular que contenía las reacciones de RT se incubó acuerdo con el siguiente protocolo:
10 25 °C durante 10 minutos 50 °C durante 30 minutos 85 °C a los 5 minutos Enfriar en hielo
15 De forma análoga al ejemplo 1, se analizaron 2 l de cDNA en una reacción de PCR de 10 l utilizando la premezcla maestra de LightCycler 480 SYBR Green I. Se realizó la PCR en tiempo real midiendo los genes ACTB, GAPDH, RPLP0 y HPRT1 con cebadores apropiados (TATAA Biocenter, panel génico de control endógeno) en un instrumento de PCR en tiempo real LightCycler 480. Los valores de Ct obtenidos se muestran en la tabla 4.
- Nombre de la muestra
- ACTB-Ct GAPDH-Ct RPLP0-Ct HPRT1-Ct
- Pocillo 1
- 21,0 19,83 21,71 24,74
- Pocillo 2
- 21,71 21,63 23,57 26,09
- Pocillo 3
- 21,19 20,95 22,86 25,88
- Pocillo 4
- 21,8 20,94 23,2 25,7
- Pocillo 5
- 23,03 22,0 24,49 26,7
- NoRT
- 40,0 N/A 40,0 N/A
- 20
- Los valores de Ct obtenidos muy similares para los quintuplicados muestran el rendimiento superior del método de la invención independientemente del tipo de célula analizada. El rendimiento también se demuestra en la Fig. 4, que muestra las curvas de amplificación y de fusión obtenidas para el gen ACTB.
- 25
10
Claims (1)
-
imagen1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP08013816 | 2008-08-01 | ||
| EP08013816 | 2008-08-01 | ||
| PCT/EP2009/005516 WO2010012464A1 (en) | 2008-08-01 | 2009-07-30 | Improved lysis and reverse transcription for mrna quantification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2539856T3 true ES2539856T3 (es) | 2015-07-06 |
Family
ID=39884115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES09802454.0T Active ES2539856T3 (es) | 2008-08-01 | 2009-07-30 | Lisis mejorada y transcripción reversa para la cuantificación de mRNA |
Country Status (7)
| Country | Link |
|---|---|
| US (2) | US8623602B2 (es) |
| EP (1) | EP2310527B1 (es) |
| JP (1) | JP5367078B2 (es) |
| CN (1) | CN102112631A (es) |
| CA (1) | CA2730761C (es) |
| ES (1) | ES2539856T3 (es) |
| WO (1) | WO2010012464A1 (es) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008135197A1 (en) * | 2007-05-03 | 2008-11-13 | Roche Diagnostics Gmbh | Improved lysis and reverse transcription for mrna quantification |
| CN103328654B (zh) | 2010-10-27 | 2017-07-11 | 哈佛学院院长等 | 立足点引物双链体的组合物及其使用方法 |
| DE102015017080B3 (de) | 2014-02-28 | 2024-01-04 | Gen-Probe Incorporated | Verfahren zur Isolierung von Nukleinsäure von zytologischen Proben in flüssigen Konservierungsmitteln, die Formaldehyd enthalten |
| EP3250716B1 (en) * | 2015-01-30 | 2021-07-07 | President and Fellows of Harvard College | Microscope-free imaging |
| US11266730B2 (en) | 2015-09-29 | 2022-03-08 | The General Hospital Corporation | Methods of treating and diagnosing disease using biomarkers for BCG therapy |
| JP2017209036A (ja) * | 2016-05-24 | 2017-11-30 | 東洋紡株式会社 | 改良されたウイルス検出方法 |
| EP3568492B1 (en) | 2017-01-10 | 2025-01-01 | President and Fellows of Harvard College | Multiplexed signal amplification |
| CN106867993A (zh) * | 2017-02-17 | 2017-06-20 | 唐旌生物科技（上海）有限公司 | 一种逆转录方法、逆转录试剂盒及其应用 |
| CN111630179B (zh) | 2018-01-26 | 2024-07-19 | 哈佛学院院长及董事 | 邻近检测方法和组合物 |
| GB201803654D0 (en) | 2018-03-07 | 2018-04-25 | Arcticzymes As | Thermolabile proteinases |
| US20220154180A1 (en) | 2019-03-13 | 2022-05-19 | Toyobo Co., Ltd. | Production and amplification of nucleic acids |
| JP2021052598A (ja) * | 2019-09-27 | 2021-04-08 | 東洋紡株式会社 | 非特異的な核酸増幅を抑制する方法 |
| CN113564159A (zh) * | 2021-07-22 | 2021-10-29 | 山东大学 | 一种分子信标探针、试剂盒及其体外转录荧光定量实时示踪检测方法和应用 |
| US20230416724A1 (en) * | 2022-06-24 | 2023-12-28 | Purigen Biosystems, Inc. | Lysis Buffers Comprising Cyanate or Thiocyanate and a Detergent |
| EP4306651A3 (en) * | 2022-07-10 | 2024-07-03 | Vilnius University | Composition and the use of cell lysis reagents |
| EP4656735A1 (en) | 2023-01-26 | 2025-12-03 | Toyobo Co., Ltd. | Inibhition of reverse transcription derived from ribosomal rna |
Family Cites Families (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5310652A (en) * | 1986-08-22 | 1994-05-10 | Hoffman-La Roche Inc. | Reverse transcription with thermostable DNA polymerase-high temperature reverse transcription |
| US5118801A (en) | 1988-09-30 | 1992-06-02 | The Public Health Research Institute | Nucleic acid process containing improved molecular switch |
| NL8900725A (nl) | 1989-03-23 | 1990-10-16 | Az Univ Amsterdam | Werkwijze en combinatie van middelen voor het isoleren van nucleinezuur. |
| US5210015A (en) | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
| JPH06508029A (ja) * | 1991-05-17 | 1994-09-14 | カイロン コーポレイション | ＮＦ−↓ｋＢ転写活性化物質のインヒビター及びその使用 |
| US5538848A (en) | 1994-11-16 | 1996-07-23 | Applied Biosystems Division, Perkin-Elmer Corp. | Method for detecting nucleic acid amplification using self-quenching fluorescence probe |
| WO1996039536A1 (en) * | 1995-06-06 | 1996-12-12 | T Cell Diagnostics, Inc. | UNIVERSAL CHEMISTRY ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF mRNA EXPRESSION |
| DE69739357D1 (de) | 1996-06-04 | 2009-05-28 | Univ Utah Res Found | Überwachung der hybridisierung während pcr |
| ES2215230T3 (es) | 1996-06-04 | 2004-10-01 | University Of Utah Research Foundation | Sistema y metodo para llevar a cabo y supervisar reacciones de cadena de polimerasa. |
| GB9716664D0 (en) * | 1997-08-06 | 1997-10-15 | Norwegian Inst Of Fisheries & | A method of removing nucleic acid contamination reactions |
| US6248535B1 (en) * | 1999-12-20 | 2001-06-19 | University Of Southern California | Method for isolation of RNA from formalin-fixed paraffin-embedded tissue specimens |
| EP2290098A1 (en) * | 2002-01-28 | 2011-03-02 | Ambion, Inc. | Preparing crude biological extracts using protease, suitable for preparing cDNA |
| EP1631685B1 (en) | 2003-05-19 | 2010-12-29 | Brandeis University | Nucleic acid processing methods, kits and devices |
| JP3714940B2 (ja) * | 2003-11-07 | 2005-11-09 | 株式会社日立ハイテクノロジーズ | Ｒｎａ抽出方法、および生体材料の分析方法 |
| MXPA05001815A (es) * | 2004-02-20 | 2005-08-24 | Hoffmann La Roche | Adsorcion de acidos nucleicos a una fase solida. |
| US20050208501A1 (en) * | 2004-03-16 | 2005-09-22 | Ambion, Inc. | Process and reagents for extraction of RNA from fractionated blood leukocytes |
| DE102004026744A1 (de) * | 2004-05-28 | 2005-12-29 | Philipps-Universität Marburg | Erfindung betreffend cDNA-Herstellung aus Zellen nach Laser-Mikrodissektion |
| EP1707623A1 (de) * | 2005-04-01 | 2006-10-04 | Qiagen GmbH | Reverse Transkription und Amplifikation von RNA bei simultaner Degradierung von DNA |
| US8309303B2 (en) * | 2005-04-01 | 2012-11-13 | Qiagen Gmbh | Reverse transcription and amplification of RNA with simultaneous degradation of DNA |
| US20060286557A1 (en) * | 2005-06-15 | 2006-12-21 | Basehore Lee S | Combined lysis and PCR buffer |
| EP1988654A1 (en) | 2007-05-02 | 2008-11-05 | Matsushita Electric Industrial Co., Ltd. | Communication scheme for channel quality information |
| WO2008135197A1 (en) | 2007-05-03 | 2008-11-13 | Roche Diagnostics Gmbh | Improved lysis and reverse transcription for mrna quantification |
-
2009
- 2009-07-30 CA CA2730761A patent/CA2730761C/en active Active
- 2009-07-30 JP JP2011520376A patent/JP5367078B2/ja active Active
- 2009-07-30 CN CN2009801304054A patent/CN102112631A/zh active Pending
- 2009-07-30 EP EP09802454.0A patent/EP2310527B1/en active Active
- 2009-07-30 WO PCT/EP2009/005516 patent/WO2010012464A1/en not_active Ceased
- 2009-07-30 ES ES09802454.0T patent/ES2539856T3/es active Active
-
2011
- 2011-01-19 US US13/008,949 patent/US8623602B2/en active Active
-
2013
- 2013-12-05 US US14/097,694 patent/US9714448B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102112631A (zh) | 2011-06-29 |
| WO2010012464A1 (en) | 2010-02-04 |
| US9714448B2 (en) | 2017-07-25 |
| US20140212958A1 (en) | 2014-07-31 |
| EP2310527A1 (en) | 2011-04-20 |
| US8623602B2 (en) | 2014-01-07 |
| JP2011528914A (ja) | 2011-12-01 |
| US20110111463A1 (en) | 2011-05-12 |
| CA2730761A1 (en) | 2010-02-04 |
| CA2730761C (en) | 2016-04-19 |
| EP2310527B1 (en) | 2015-04-29 |
| JP5367078B2 (ja) | 2013-12-11 |
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