Navigation Links
Efficient and Reliable Linear,,, Amplification of cRNA

Gene Expression Profiling Using Microarray Kits from Roche Applied Science

The use of gene-expression profiling, a genomic application measuring mRNA transcript levels of many genes in parallel, has been growing rapidly over the last few years. Besides the actual microarrays and instrumentation for hybridization and imaging, target preparation is of major importance for reliability and sensitivity of expression-profiling experiments. Several different target preparation strategies have been recently developed that are mainly related to the amount of starting material available.


Benefits of the Roche Applied Science Microarray Kits

  • High yield and integrity of labeled cRNA

  • Representational amplification of small amounts of RNA for gene-expression analysis

  • Seamless workflow from cDNA synthesis to cRNA labeling

  • Fast and easy procedure

  • Incorporation of hapten- or fluorescence-labeled nucleotides

  • Only one labeled nucleotide needed

  • Compatible with spotted or in situ synthesized microarrays

Target Preparation Workflow
Especially in medical research applications, sample material is often limited and therefore target amplification is mandatory. Starting from a few micrograms of total RNA, linear amplification of cRNA utilizing T7 RNA polymerase for the preparation of the labeled target, first described by van Gelder and Eberwine [1], has become a standard procedure (Figure 1 a). This protocol consists of a first- and second-strand cDNA synthesis followed by cRNA in vitro transcription using T7 RNA polymerase. For first-strand cDNA synthesis an oligo(dT) primer linked to the T7 promoter sequence is used. In the next step, this T7 linker allows the linear amplification of the template and the simultaneous incorporation of labeled nucleotides with T7 RNA polymerase, generating singlestranded, labeled cRNA. The complete target-preparation workflow consists of multiple enzymatic steps determining the quality and integrity of the targets.

Experimental Validation

To validate the T7 RNA polymerase-based target synthesis procedure, the Roche Applied Science (RAS) Microarray RNA Target Synthesis Kit (T7) and the kit of a major Supplier (A) were tested on three different human acute myeloid leukemia (AML) research samples and one chronic lymphocytic leukemia (CLL) research sample (Figure 1b).

First, 5 g of total RNA was converted into double-stranded (ds) cDNA using the RAS Microarray cDNA Synthesis Kit (eight independent reactions). One group of ds cDNA samples was purified with the RAS Microarray Target Purification Kit, then amplified with T7 RNA polymerase and labeled with Biotin-UTP, using the RAS Microarray RNA Target Synthesis Kit (T7). The resulting labeled cRNA was purified with the RAS Microarray Target Purification Kit. The ds cDNA of the other sample group was purified according to the Supplier As protocol, followed by in vitro transcription and labeling with Biotin- UTP and Biotin-CTP using Supplier As kit. Purification of the labeled cRNA was carried out according to Supplier As recommendations. For all samples, 10 g labeled cRNA was hybridized to HG-U133A GeneChip Arrays. To estimate the performance of the T7 amplification, the yield of biotin-labeled cRNA and the hybridization results obtained from HG-U133A GeneChip Arrays were analyzed (Table 1).

Fast Procedure with High Efficiency

The yield of labeled cRNA is slightly lower for three out of four samples using the RAS Microarray Kits. However, both reaction volume (20 l vs. 40 l) and reaction time (2 hours vs. 4 hours) are reduced by half with the RAS Microarray RNA Target Synthesis Kit (T7) compared to the approach of Supplier A, indicating the higher efficiency and swifter procedure provided by the kit. In addition, the RAS Microarray Target Purification Kit simplifies the cDNA/cRNA purification steps of the Eberwine protocol.

The hybridization data obtained from the HG-U133A GeneChip Arrays show a higher number of present calls for the labeled cRNA that was generated with the RAS Microarray RNA Target Synthesis Kit (T7), signifying increased sensitivity. The kit protocol is economically optimized for the incorporation of only one labeled nucleotide (e.g., Biotin-UTP), and our results indicate that there is no further improvement of sensitivity by additional labeled nucleotides.

Furthermore, the 3′ 5′ ratio of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) displays a lower ratio for these targets, demonstrating the larger amount and integrity of full-length cRNA targets achieved when using the RAS Microarray RNA Target Synthesis Kit (T7) for linear amplification and labeling.

The use of the RAS Microarray Kits demonstrates a more efficient target preparation procedure that results in a greater integrity of labeled target cRNA.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
2. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini
3. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
4. Efficient Transfection of Neurospora Crassa
5. Efficient Transfection of Neurospora Crassa
6. Efficient Recovery of Ultrapure Plasmid DNA
7. Efficient and Reliable PCR Setup Using Eppendorf MasterMix
8. Efficient DNA transfection of primary CNS neurons using TransMessenger Transfection Reagent
9. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
10. Cloning Based on Efficient Three-Fragment Assembly DNA Ligation
11. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Efficient and Reliable Linear Amplification cRNA

(Date:8/3/2015)... , August 3, 2015 ... Study on 3D Cell Culture: Asia ... Persistence Market Research, the global 3D cell culture market was valued ... to expand at a CAGR of 29.1% to account ... Full Report with TOC:  http://www.persistencemarketresearch.com/market-research/3d-cell-cultures-market.asp 3D ...
(Date:7/31/2015)... China Cord Blood Corporation (NYSE: CO) ("CCBC" ... provider of cord blood collection, laboratory testing, hematopoietic stem ... that the Company has filed its Annual Report on ... The filed Form 20-F includes audited financial statements for ... 20-F can be accessed by visiting the U.S. Securities ...
(Date:7/30/2015)... ... July 30, 2015 , ... Tronics, ... as Sales & Marketing Director. , With more than 25 years of ... for reinforcing Tronics’ business development activities worldwide. He brings to the company his ...
(Date:7/30/2015)... July 30, 2015   Senomyx , Inc. (NASDAQ: ... science technologies to discover, develop, and commercialize novel flavor ... today reported financial results for the second quarter 2015. ... 2015, we remain on track to achieve our commercial ... Executive Officer of the Company. "Since our last quarterly ...
Breaking Biology Technology:Global 3D Cell Culture Market Will Reach US$ 2,717.6 Million by 2020: Persistence Market Research 2Global 3D Cell Culture Market Will Reach US$ 2,717.6 Million by 2020: Persistence Market Research 3Global 3D Cell Culture Market Will Reach US$ 2,717.6 Million by 2020: Persistence Market Research 4China Cord Blood Corporation Files Its Annual Report on Form 20-F 2Tronics appoints Francois Vieillard as Sales and Marketing Director 2SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 2SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 4SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 5SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 6SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 7SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 8SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 9
... PARK, N.C., June 14, 2011 Bayer CropScience ( ... top innovative crop science companies in the areas of ... announced the release of a video featuring the organization,s ... award-winning program, Making Science Make Sense (MSMS) . ...
... PAREXEL International Corporation (NASDAQ: PRXL ... Wells Fargo Healthcare Conference in Boston, MA.  James Winschel, Sr. ... presentation on PAREXEL at 2:00 p.m. ET on Wednesday, June ... will be available through the "Investors" section of ...
... industry delivered solid top- and bottom-line growth in 2010, with ... a row. Yet funding for research and ... firms in the sector, which tend to be pre-commercial stage ... development. This has placed new pressure on the traditional biotech ...
Cached Biology Technology:Bayer CropScience Releases Video About Making Science Make Sense Program At Durham Performance Learning Center 2Bayer CropScience Releases Video About Making Science Make Sense Program At Durham Performance Learning Center 3Bayer CropScience Releases Video About Making Science Make Sense Program At Durham Performance Learning Center 4Bayer CropScience Releases Video About Making Science Make Sense Program At Durham Performance Learning Center 5PAREXEL International to Present at Wells Fargo Healthcare Conference 2Despite Renewed Growth in 2010, Biotech Industry Faces R&D Challenges 2Despite Renewed Growth in 2010, Biotech Industry Faces R&D Challenges 3Despite Renewed Growth in 2010, Biotech Industry Faces R&D Challenges 4Despite Renewed Growth in 2010, Biotech Industry Faces R&D Challenges 5Despite Renewed Growth in 2010, Biotech Industry Faces R&D Challenges 6
(Date:7/7/2015)... , July 7, 2015 NXT-ID, Inc. (NASDAQ: ... on the growing mobile commerce market announces a revised version of ... week. The commercial will air on CNBC in New ... Angeles and San Francisco metro ... commercial focuses on Wocket,s ability to replace all the cards in ...
(Date:7/2/2015)... Research and Markets( http://www.researchandmarkets.com/research/jvft55/next_generation ) has ... Market by Application, Technology, Function & Geography - Global ... The next generation biometrics market is expected to ... 17.9% between 2015 and 2020 Travel & ... Safran SA ( France ), NEC ...
(Date:6/30/2015)... -- To bolster its efforts and commitment to fighting cyber ... addition of two new team members. ... David Raviv will act as head of strategic alliances. ... providing the most secure solutions for the enterprise market. ... Technologies, a provider of security and management products, which was ...
Breaking Biology News(10 mins):Wocket Smart Wallet Commercials Airing on CNBC to Showcase This Summer's Must- Have Tech Product 2Wocket Smart Wallet Commercials Airing on CNBC to Showcase This Summer's Must- Have Tech Product 3Next Generation Biometrics Market 2015 - Global Forecast to 2020 with Safran, NEC, 3M Cogent, Fujitsu & Suprema Dominating the $24 Billion Market 2Next Generation Biometrics Market 2015 - Global Forecast to 2020 with Safran, NEC, 3M Cogent, Fujitsu & Suprema Dominating the $24 Billion Market 3HYPR Corp. Expands Biometric Authentication Team with Enterprise All Stars 2HYPR Corp. Expands Biometric Authentication Team with Enterprise All Stars 3
... This release is available in French . ... in collaboration with Nova Scotia Agricultural College, have discovered what ... double the lifespan of Christmas trees in homes. The authors ... journal Trees . The researchers have identified a ...
... is not just a feature unique to humans and ... revealed that also brown trout have individual characters and ... Department of Zoology at the University of Gothenburg has ... from watercourses on the west coast of Sweden. ...
... of Change, literally took 100 years to write. The first ... It was repeated from 1956-1958 and a technical book was ... that first survey was approaching, ornithologists Mike Ward, Jeff Walk, ... to do it again and write a book, this time ...
Cached Biology News:Researchers discover a way to delay Christmas tree needle loss 2New book on 100 years of Illinois birds 2New book on 100 years of Illinois birds 3New book on 100 years of Illinois birds 4
Human beta IG-H3 MAb (Clone 348506) Keywords: BIGH3, betaIG-H3 Protein Family: Extracellular Matrix Molecules...
Human Cystatin F MAb (Clone 292103)...
Rat CNTF R alpha Affinity Purified Polyclonal Ab...
... an inhibitor of caspase-3/7 ... of inhibitor required to ... be determined empirically for ... inhibitor is sufficient to ...
Biology Products: