Navigation Links
Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue

RecoverAll Total Nucleic Acid Isolation Kit
For decades, FFPE tissue has been the staple of histologists and pathologists for creating tissue slides and making patient diagnoses. More than 400 million FFPE tissue samples exist for cancer studies alone, and many of these samples can be tied to clinical data. Using one simple kit, the RecoverAll Total Nucleic Acid Isolation Kit (patent pending), researchers can now isolate total nucleic acids (DNA and RNA, including miRNA) from the same FFPE sample. Efficient protease digestion conditions allow RNA isolation in less than one day. The nucleic acids recovered are ideal for various downstream applications such as microarray analysis, qRT-PCR, and mutation screening.


Nucleic Acid Isolation from FFPE Tissue

Ambion has developed a new method for isolating nucleic acids from human and mouse FFPE tissue. Up to four 20 m sections or unsectioned core samples <35 mg can be processed per reaction. Following deparaffinization and a rigorous protease digestion, the sample can be split into separate aliquots for isolation of total RNA (including miRNA) and genomic DNA using an optimized glass-fiber filter purification protocol.


Total RNA Isolation from FFPE Samples

Although the RecoverAll Kit cannot reverse RNA fragmentation that may have already occurred (see sidebar, Challenges of Molecular Analysis of FFPE Tissues), the protease digestion conditions are designed to release a maximal amount of RNA of all sizes in a significantly shorter period of time (3 hr) compared to currently available methods. Recovery of mRNA has been verified by qRT-PCR (see below), and microRNAs have been documented with both Northern blot and array analysis (see Examine MicroRNA Profiles from Archived FFPE Tissue).


Gene Expression from FFPE Samples

Figure 1 shows the results of several real-time RT-PCR experiments comparing detection of a variety of genes from fixed versus frozen mouse brain samples. The Ct values from an equal mass of the FFPE samples were higher than those from the frozen controls, indicating that other modifications from the fixation process have adversely affected the RNA as a reverse transcription template.

Figure 1. Ct Values from Real-Time RT-PCR of Frozen and FFPE Mouse Brain. Half of each of four mouse brain samples were flash frozen in liquid nitrogen then stored at 80C; the other half was fixed and embedded using a standard hospital protocol. RNA was isolated from one 20 m slice from each FFPE mouse brain using the RecoverAll Total Nucleic Acid Isolation Kit. RNA from the frozen controls was isolated using the mirVana miRNA Isolation Kit. RNA (400 ng) from each sample was used in two-step real-time RT-PCR. cDNA was synthesized using the RETROscript Kit. Random decamer primers, one tenth of the RT reaction, and SuperTaq Polymerase were used for PCR. Each bar represents the mean standard deviation for 8 replicates. TBP=TATA Binding Protein; GAPDH=Glyceraldehyde-3-phosphate Dehydrogenase; UBC=Ubiquitin C; PKC=Protein Kinase C; Recc1=Replication Factor C; RNAPol II=RNA Polymerase II; FAS=Fatty Acid Synthase; DDPK=DNA Activated Protein Kinase.


DNA Isolation from FFPE Tissues

Researchers can now recover DNA from FFPE samples just as easily as RNA by simply increasing the digestion time to 2 days. Isolation of DNA includes an RNase treatment to fully eliminate RNA. Figure 2 shows an Agilent 2100 bioanalyzer scan of total nucleic acids, RNA, and DNA from an FFPE sample processed with the RecoverAll Kit.

Figure 2. Agilent 2100 bioanalyzer Scan Demonstrating Presence of RNA and DNA from FFPE Mouse Liver. Nucleic acids were isolated from FFPE mouse liver (20 m section, fixed and embedded using standard hospital protocol) using the RecoverAll Total Nucleic Acid Isolation Kit. Three equal amounts of sample (based on A260) were treated as follows: untreated control received no DNase or RNase treatment; RNA was isolated by DNase treatment of the nucleic acid sample; DNA was isolated by RNase treatment of the nucleic acid sample. An equal volume of each was analyzed on the Agilent 2100 bioanalyzer.


Get More Data from Your FFPE Samples

Many researchers considered their archives of FFPE tissues lost to molecular analysis. Now, with Ambion's RecoverAll Kit, you can isolate the full range of nucleic acids from a single FFPE tissue sample.


Scientific Contributors

Rick Conrad, Tim Barta, Emily Zeringer Ambion, Inc.


back to top


Ordering Information
Cat# Product Name Size 1710 RETROscript Kit 40 rxns 1975 RecoverAll Total Nucleic Acid Isolation Kit for FFPE 40 purifications 2054 SuperTaq Plus Polymerase (Cloned) 5 U/l 50 U 2056 SuperTaq Plus Polymerase (Cloned) 5 U/l 250 U
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Recovery of DNA from LMP Agarose1
2. Efficient Recovery of Ultrapure Plasmid DNA
3. Improve Recovery and Reproducibility with Pre-Treatment of Analytical TSK-GEL G3000SWXL Size Exclusion Columns
4. Yeast Protein Production System Features High Yields and One-Step Purification
5. ArrayScript RT: Maximize Yields of Full-length cDNA
6. Synthesize High Yields of Biotinylated aRNA
7. Translate High Yields of Active Protein
8. Translate High Yields of Biologically Active Proteins
9. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
10. High-Throughput Isolation of Total RNA
11. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
Post Your Comments:
(Date:8/1/2014)... August 01, 2014 This is a ... the Ammonium Bicarbonate industry in Global and China. The ... definitions, applications and industry chain structure. Global market analysis ... focus on history, developments, trends and competitive landscape of ... situation is also offered. , The report also focuses ...
(Date:8/1/2014)... CHICAGO , Aug. 1, 2014 AACC, ... better health through laboratory medicine, unveiled an updated brand ... laboratory professionals adapt to the changing healthcare environment and ... insight to physicians, so patients get the care they ... make up AACC,s membership play an essential role in ...
(Date:8/1/2014)... Aug. 1, 2014 Research and Markets ... Single Nucleotide Polymorphism (SNP) Genotyping and Analysis Market 2014-2018" ... http://photos.prnewswire.com/prnh/20130307/600769 Single Nucleotide Polymorphism (SNP) ... of SNPs in the genome of various species. SNPs ... genotyping and analysis technology can analyze thousands of SNPs ...
(Date:8/1/2014)... Glencoe Software, Inc. is pleased ... ( http://glencoesoftware.com/data-inpress.html ), a suite of data ... into on-line publications.  The suite comprises Glencoe ... Photo - http://photos.prnewswire.com/prnh/20140731/132410   ... The DataViewer is a browser-based ...
Breaking Biology Technology:Ammonium Bicarbonate Market in Internations & Chinese Regions Research Analysis Now Available at DeepResearchReports.com 2Ammonium Bicarbonate Market in Internations & Chinese Regions Research Analysis Now Available at DeepResearchReports.com 3AACC Debuts Refreshed Brand Identity 2Global Single Nucleotide Polymorphism (SNP) Genotyping and Analysis Market 2014-2018 2Glencoe Software Launches Data InPress 2Glencoe Software Launches Data InPress 3
... Richard Thieme's new book, Islands in the Clickstream, is a ... in May, Thieme's book has been hailed as extremely insightful ... world of technology and present his thoughts in the context ... in chief of Information Security Magazine, said: This book is ...
... associated with television commercials offering things such as accredited ... For many years, distance education was associated with ... locksmithing and gunsmithing, all earned over the mail, from ... Business has been at the forefront of utilizing ...
... they either dont understand it or they dont feel the ... value of networking and when they should network. , ,For ... (such as a new job or a product to sell). ... a process over time. If done effectively, their needs will ...
Cached Biology Technology:Technology isn't about technology 2Technology isn't about technology 3Technology isn't about technology 4Charter Business powers distance learning 2Charter Business powers distance learning 3Charter Business powers distance learning 4Dispelling the top five myths about attending networking events 2Dispelling the top five myths about attending networking events 3Dispelling the top five myths about attending networking events 4
(Date:7/31/2014)... MANHATTAN Kansas State University researchers have completed ... of burning Flint Hills prairie at different times ... of the current late spring time frame has ... in fact, may have multiple benefits. , The ... and the Konza Prairie Biological Station fire chief, ...
(Date:7/31/2014)... of animal models against the highly infectious and virulent ... disease that kills approximately 30,000 Americans annually. The research ... Immunity . , In the study, the vaccine ... toxins produced by C. difficile , as well ... that mimics the human disease, after only two immunizations. ...
(Date:7/31/2014)... the photovoltaic effect in 1839, humankind has sought to ... its own purposes. In a new research report published ... FASEB Journal , scientists may have uncovered a ... focusing on a naturally occurring combination of lipids that ... over time and across speciessuggests that this specific natural ...
Breaking Biology News(10 mins):Study finds benefits to burning Flint Hills prairie in fall and winter 2Study finds benefits to burning Flint Hills prairie in fall and winter 3C. difficile vaccine proves safe, 100 percent effective in animal models 2Scientists shine bright new light on how living things capture energy from the sun 2
... Drosophila mauritiana , a close relative of the well ... around our fruit bowls in summer. Nolte and colleagues ... the various genes it contains. The information will naturally ... this organism. But the present study goes much, much ...
... wet and dry foods, cats are able to select ... a consistent intake of protein, fat and carbohydrate, i.e. ... of Comparative Physiology B , the research shows that ... selection despite differences in the macronutrient content, moisture level ...
... MD, PhD, Director of the Center for Cell Engineering at ... the effectiveness of a new technique that could allow the ... were reported online today in Nature Biotechnology . ... cells that have been augmented in the laboratory have ...
Cached Biology News:What's in a genome? 2Cats are able to navigate complex combinations of wet and dry foods to achieve a consistent intake of protein, fat and carbohydrate 2New technique could make cell-based immune therapies for cancer safer and more effective 2
... (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide), 50mg/vial. MTT is a ... living cells to yield a dark blue ... and even freshly dead cells do not ... is a component of Chemicons MTT assay ...
... of microplate readers combines reagent dispensing with ... affordable, easy-to-use platform for the complete range ... design and adaptability of the Infinite 200 ... a monochromator (M200) or filter (F200) system ...
Rabbit polyclonal to SMARCA2 / BRM - ChIP Grade ( Abpromise for all tested applications). entrezGeneID: 6595 SwissProtID: P51531...
... Glutamate receptors constitute the principal ... Two classes of glutamate receptors ... receptors. Ionotropic glutamate receptors are ... (GluR1-7, NMDA1-3, KAI-2) which comprise ...
Biology Products: