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 uti lizing 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 accor ding 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 effici ent 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:


(Date:6/24/2016)... 24, 2016  Regular discussions on a range of subjects ... the two entities said Poloz. Speaking at a ... , he pointed to the country,s inflation target, which ... "In certain areas ... have common economic goals, why not sit down and address ...
(Date:6/23/2016)... ... June 23, 2016 , ... Mosio, a leader ... “Clinical Trials Patient Recruitment and Retention Tips.” Partnering with experienced clinical research professionals, ... providing practical tips, tools, and strategies for clinical researchers. , “The landscape of ...
(Date:6/23/2016)... 2016 A person commits a crime, and the ... track the criminal down. An outbreak of foodborne ... Administration (FDA) uses DNA evidence to track down the bacteria ... far-fetched? It,s not. The FDA has increasingly used a complex, ... foodborne illnesses. Put as simply as possible, whole genome sequencing ...
(Date:6/23/2016)... -- The Biodesign Challenge (BDC), a university competition that asks ... systems and biotechnology, announced its winning teams at the ... York City . The teams, chosen ... MoMA,s Celeste Bartos Theater during the daylong summit. Keynote ... of architecture and design, and Suzanne Lee , ...
Breaking Biology Technology:
(Date:6/27/2016)... Research and Markets has announced the addition of the ... offering. The report forecasts the ... at a CAGR of 12.28% during the period 2016-2020. ... analysis with inputs from industry experts. The report covers the market ... also includes a discussion of the key vendors operating in this ...
(Date:6/22/2016)... June 22, 2016  The American College of Medical Genetics ... Executive Magazine as one of the fastest-growing trade shows ... at the Bellagio in Las Vegas . ... percentage of growth in each of the following categories: net ... and number of attendees. The 2015 ACMG Annual Meeting was ...
(Date:6/22/2016)... ANGELES , June 22, 2016 /PRNewswire/ ... identity management and verification solutions, has partnered ... edge software solutions for Visitor Management, Self-Service ... provides products that add functional enhancements ... partnership provides corporations and venues with an ...
Breaking Biology News(10 mins):