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.



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:

(Date:11/25/2015)... 2015 Studies reveal the differences ... and pave the way for more effective treatment for one ...   --> --> ... problems in cats, yet relatively little was understood about the ... have been conducted by researchers from the WALTHAM Centre for ...
(Date:11/25/2015)... DIEGO , Nov. 25, 2015  Neurocrine Biosciences, ... Kevin Gorman , President and CEO of Neurocrine Biosciences, ... Healthcare Conference in New York . ... to visit the website approximately 5 minutes prior to ... A replay of the presentation will be available on ...
(Date:11/25/2015)... IN (PRWEB) , ... November 25, 2015 , ... ... (AMA) and the Organization of Black Aerospace Professionals (OPBAP) has been formalized with ... and other AMA team leaders met with OPBAP leaders Capt. Karl Minter and ...
(Date:11/25/2015)... 2015 The Global Genomics ... professional and in-depth study on the current state ... ) , The report ... definitions, classifications, applications and industry chain structure. The ... markets including development trends, competitive landscape analysis, and ...
Breaking Biology Technology:
(Date:11/11/2015)... , Nov. 11, 2015   MedNet Solutions , ... spectrum of clinical research, is pleased to announce that it ... Clinical Trials (PCT) event, to be held November 17-19 ... able to view live demonstrations of iMedNet ... learn how iMedNet has been able to deliver ...
(Date:11/4/2015)... , November 4, 2015 ... new market report published by Transparency Market Research "Home Security ... Trends and Forecast 2015 - 2022", the global home security ... 30.3 bn by 2022. The market is estimated to ... period from 2015 to 2022. Rising security needs among ...
(Date:10/29/2015)... , Oct. 29, 2015  Rubicon Genomics, ... for U.S. distribution of its DNA library preparation ... and Rubicon,s new ThruPLEX Plasma-seq kit. ThruPLEX Plasma-seq ... the preparation of NGS libraries for liquid biopsies--the ... diagnostic and prognostic applications in cancer and other ...
Breaking Biology News(10 mins):