IP Library › Granted Patent US 6,995,020
Granted Patent B2
US 6,995,020 · App. 10/624,233 · Granted Feb 7, 2006

Methods and compositions for the preparation and use of fixed-treated cell-lines and tissue in fluorescence in situ hybridization

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Quick Facts
Patent No.
US 6,995,020
App. No.
10/624,233
Granted
Feb 7, 2006
Kind
B2
Abstract

This invention relates to methods for the detection of one or more mRNA transcripts in paraffin-embedded tissue by “mRNA liberation in fixed-treated tissue or ‘MLIFTT’”. This method includes treating the tissue with ammonia-ethanol and sodium borohydride combined with pressure cooking of the tissue. The chemical treatments reduce the tissue autofluorescence and the physical treatments overcome the interference created by the fixation-induced chemical bonds. The methods of the present invention can be utilized to identify a plurality of mRNA transcripts in a microarray format.

Claims (13)

1. A method for preparing a mammalian cell-line and/or mammalian tissue sample for in situ hybridization, comprising the steps of:

(a) pressure cooking the sample in a decloaking chamber at a temperature of about 125° C. reaching a pressure of between about 20 to about 24 PSI; and

(b) treating the pressure cooked sample with ammonia-ethanol in a concentration of about 0.25% and sodium borohydride in a concentration of about 5%.

2. The method of claim 1 wherein the sample is fixed-treated.

3. The method of claim 2 wherein the fixed-treated sample is paraffin embedded.

4. The method of claim 1 wherein the tissue or the cell is human.

5. A pressure cooked composition comprising:

a fixed-treated tissue according to claim 1 .

6. The pressure cooked composition according to claim 5 , wherein the fixed-treated tissue is paraffin embedded.

7. A method for reducing autofluorescence when performing FISH on a fixed treated tissue sample, the method comprising

(a) pressure cooking the sample in a decloaking chamber at a temperature of about 125° C. reaching a pressure of between about 20 to about 24 PSI; and

(b) treating the pressure cooked sample with ammonia-ethanol in a concentration of about 0.25% and sodium borohydride in a concentration of about 5%.

wherein both steps are performed on the sample prior to performing FISH.

Assignments (7)
THIS IS TO CORRECT ERRORS IN A COVER SHEET PREVIOUSLY RECORDED (REEL/FRAME 027502/0828): CORRECTION OF THE ASSIGNOR'S NAME TO AUREON (ASSIGNMENT FOR THE BENEFIT OF CREDITORS), LLC.; CORRECTION OF THE ASSIGNEE'S NAME TO AUREON, INC. Recorded May 22, 2012
From: AUREON (ASSIGNMENT FOR THE BENEFIT OF CREDITORS), LLC
To: AUREON, INC.
Reel/Frame 028252/0442 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 21, 2012
From: AUREON, INC.
To: CHAMPALIMAUD FOUNDATION
Reel/Frame 028244/0077 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 9, 2012
From: AUREON BIOSCIENCES, INC.
To: DAVID SANS
Reel/Frame 027502/0828 →
SECURITY AGREEMENT Recorded Oct 25, 2010
From: AUREON BIOSCIENCES, INC.
To: ATLAS VENTURE FUND VI GMBH & CO. KG; ATLAS VENTURE FUND VI, L.P.; ATLAS VENTURE ENTREPRENEURS' FUND VI, L.P.; PFIZER INC.; MINTZ LEVIN INVESTMENTS LLC; SPROUT CAPITAL IX, L.P.; DLJ CAPITAL CORP.; SPROUT ENTREPRENEURS FUND, L.P.; SPROUT IX PLAN INVESTORS, L.P.; CANTABRIA DE ACTIVOS, S.L.; CORPORACION MASAVEU, S.A.; INICIATIVAS DIGITALES; IPETEX S.A.; INVERSIONES VECU 2006, S.L.; LEALTAD DESARROLLO, S.C.R., S.A.; BASSETTI, MARCO; C+PA-CIMENTO E PRODUTOS ASSOCIADOS, S.A.
Reel/Frame 025178/0899 →
SECURITY AGREEMENT Recorded Aug 9, 2010
From: AUREON BIOSCIENCES, INC.
To: ATLAS VENTURE FUND VI, L.P.; ATLAS VENTURE FUND VI GMBH & CO. KG; ATLAS VENTURE ENTREPRENEURS' FUND VI, L.P.; SPROUT ENTREPRENEURS FUND, L.P.; SPROUT CAPITAL IX, L.P.; DLJ CAPITAL CORP.; SPROUT IX PLAN INVESTORS, L.P.; PFIZER INC; MINTZ LEVIN INVESTMENTS LLC; INICIATIVAS DIGITALES; INVERSIONES VECU 2006, S.L.; LEALTAD DESARROLLO, S.C.R., S.A.; IPETEX S.A.; CANTABRIA DE ACTIVOS, S.L.; CORPORACION MASAVEU, S.A.; PEREZ DE ARMINAN, ALFREDO; FERNANDEZ DE VILLAVICENCIO, GABRIEL STAMOGLOU; FERNANDEZ DE VILLAVICENCIO, ALEJANDRO STAMOGLOU
Reel/Frame 024946/0493 →
SECURITY AGREEMENT Recorded Jan 12, 2010
From: AUREON LABORATORIES, INC.
To: ATLAS VENTURE ENTREPRENEURS' FUND VI, L.P.; ATLAS VENTURE FUND VI GMBH & CO. KG; ATLAS VENTURE FUND VI, L.P.; SPROUT CAPITAL IX, L.P.; DLJ CAPITAL CORP.; SPROUT IX PLAN INVESTORS, L.P.; SPROUT ENTREPRENEURS' FUND, L.P.; PFIZER INC.; CANTABRIA DE ACTIVOS, S.L.; CORPORACION MASAVEU, S.A.; LEALTAD DESARROLLO, S.C.R., S.A.; INICIATIVAS DIGITALES; INVERSIONES VECU 2006, S.L.; MARCO BASSETTI; ESTRELA - SGPS, S.A.; MINTZ LEVIN INVESTMENTS LLC
Reel/Frame 023768/0486 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 29, 2005
From: CAPODIECI, PAOLA; EDELSON, JON
To: AUREON LABORATORIES, INC.
Reel/Frame 017283/0643 →