Navigation Links
Perform Microarray Analysis with Limited Bacterial RNA


Linear RNA amplification technology for bacterial gene expression analyses
As little as 10 ng of mRNA or 100 ng total RNA required for array analysis
Low input requirements enable analysis of mixed host-pathogen samples
Full-length cDNA yields maximized by ArrayScript RT
Highly reproducible and sensitive

Over the past decade, analysis of whole-genome expression profiling with DNA microarrays has revolutionized the way biologists study gene function. RNA amplification has become the preferred method of preparing probes for most microarray platforms. Unfortunately, the benefits of RNA amplification have not been easily accessible to researchers analyzing bacterial gene expression profiles. The absence of poly(A) tails on bacterial mRNA prevents the use of oligo(dT) primed reverse transcription, which is at the heart of most amplification methods. The lack of an appropriate method for bacterial RNA amplification has hampered studies that rely on rare or limited samples, such as environmental collections, small volume cultures, and bacterial RNA isolated from host cells. With over 160 microbial genomes fully sequenced, and many more in progress, an unprecedented opportunity to perform global expression analysis on important pathogens and model organisms is now available.


MessageAmp II-Bacteria Kit
MessageAmp II-Bacteria (patent pending) is based on a linear RNA amplification technology that enables whole genome expression analysis from limited bacterial samples. By amplifying RNA, microarray analyses can now be performed with up to 500-fold less starting material. MessageAmp II-Bacteria can be used with any bacterial species and is compatible with purified total RNA, bacterial mRNA enriched using Ambions MICROBExpress Kit (see sidebar), or bacterial RNA enriched from host cell mixtures with the MICROBEnrich Kit (see sidebar). As little as 10 ng of enriched bacterial mRNA or 100 ng of total RNA can be amplified to yield sufficient antisense amplified RNA (aRNA) for microarray analysis.


Linear Bacterial RNA Amplification
MessageAmp II-Bacteria is based on a linear amplification method (Figure 1) in which the RNA sample is first polyadenylated with Poly(A) Polymerase, then reverse transcribed using oligo(dT) primers containing a T7 RNA polymerase promoter. The resulting cDNA is used to produce aRNA by in vitro transcription (IVT) primed by the added T7 promoter. The aRNA can be labeled during IVT with a variety of modified nucleotides (for glass slide microarrays or Affymetrix GeneChip arrays), or it can be used to generate 33P-labeled sense strand cDNA (for nylon PCR arrays).

Figure 1. Schematic of MessageAmp II-Bacteria Procedure.


Yields of Amplified RNA
It is important to understand that the amplified RNA yields obtained with the MessageAmp II-Bacteria Kit or any amplification system will vary based on factors such as RNA isolation method, input RNA amount, RNA quality, the bacterial species from which the RNA was isolated, and incubation time of the in vitro transcription reaction. Total RNA isolation methods that include a glass fiber filter based purification step, such as Ambions RiboPure-Bacteria Kit, give higher aRNA yields than organic extraction methods. RNA purity and intactness can also have a significant impact on amplification. Figure 2 shows typical aRNA yields for 5 bacterial species using 100 ng of total RNA isolated with the RiboPure-Bacteria Kit and amplified using MessageAmp II-Bacteria and a 6 hour IVT reaction. Even with a short 6 hour IVT incubation time, 100 ng of input total RNA results in sufficient aRNA for several replicate array experiments. A 14 hour IVT incubation is recommended when <100 ng of input RNA is used.

Figure 2. MessageAmp II-Bacteria aRNA Yields. MessageAmp II-Bacteria yields of aRNA (g) for 5 different bacterial species. Yields are averages from triplicate, 6 hour in vitro transcription reactions using 100 ng total RNA. E. coli was tested with both 100 ng and 10 ng total RNA input. aRNA concentrations were measured by UV absorbance.


Whole Genome Microarray Analysis
Affymetrix E. coli Antisense Whole Genome Arrays were used to compare biotinylated MessageAmp II-Bacteria aRNA derived from E. coli total RNA and MICROBExpress enriched mRNA, as well as the recommended Affymetrix labeling protocol using 10 g E. coli total RNA. The replicate GeneChip arrays shown in Figure 3 demonstrate high reproducibility for the MessageAmp II-Bacteria technology and the Affymetrix recommended labeling protocol. Pearsons Correlation Coefficients for all technical replicate comparisons was >0.995. The sensitivity (% Present Calls) obtained with MessageAmp II-Bacteria is comparable to the standard Affymetrix labeling protocol, as is the call concordance between replicates. (See article in previous issue for a more complete definition of sensitivity and concordance analysis.) Thus, MessageAmp II-Bacteria enables you to use significantly less starting material without compromising the quality of microarray data.

Figure 3. Affymetrix E. coli Antisense Genome Array Replicate Analysis. Scatter plots of log signal for replicate labeling reactions using Affymetrix E. coli antisense whole genome arrays (all 7312 probe sets are shown). Either E. coli total RNA or enriched mRNA derived from it (MICROBExpress Kit) was used with the recommended Affymetrix labeling protocol or Ambions MessageAmp II-Bacteria biotin IVT labeling protocol. The data was analyzed with Affymetrix Microarray Suite, Version 5 with scaling to all probe sets and a target signal value of 500. 2, 3, 10 and 30-fold change lines are represented.


Tips for Bacterial RNA Amplification
When using MessageAmp II-Bacteria for expression analysis, Ambion recommends that the researcher use similar input RNA mass amounts, RNA type (total RNA or mRNA), and IVT incubation times when performing comparisons between samples. Also, as with other RNA amplification methods, only amplified samples should be compared to one another. Unamplified samples should not be compared directly with amplified samples. All amplification methods, or any labeling method for that matter, produce some biases in mRNA representation. What is important for expression analysis is that the biases are reproducible within a method and that similar labeling methods are used on samples being compared. MessageAmp II-Bacteria compares favorably with standard labeling protocols in terms of reproducibility and dynamic range, but with a dramatically reduced requirement for input RNA.


A Complete Kit for Bacterial RNA Amplification
The MessageAmp II-Bacteria Kit provides reagents for 20 RNA amplifications starting with as little as 10 ng bacterial mRNA or as much as 500 ng total RNA. A variety of labeling methods can be used with the kit, including 33P cDNA labeling, direct labeling with Cy dyes or biotin, and indirect coupling using aminoallyl nucleotides (modified nucleotides are not provided with the kit). E. coli total RNA is provided as a control.

While MessageAmp II-Bacteria can be successfully used with total RNA, MICROBExpress enriched bacterial mRNA is a much better choice for microarray analysis. A 10-fold to 100-fold increase in sensitivity is seen when using enriched mRNA, compared with total RNA. Overall signal and signal-to-noise ratios can be dramatically increased by using RNA prepared using the MICROBEnrich and/or MICROBExpress Kits with MessageAmp II-Bacteria [1].


back to top


Ordering Information
Cat# Product Name Size 1790 MessageAmp II-Bacteria RNA Amplification Kit 20 rxns 1901 MICROBEnrich Kit 20 purifications 1905 MICROBExpress Bacterial mRNA Enrichment Kit 20 rxns 1925 RiboPure-Bacteria Kit 50 purifications
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase
2. Comparing Fidelity and Performance of Proofreading PCR Enzymes
3. PCR Performance Comparisons Between pfuturbo and Taq DNA Polymerases
4. pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning
5. Performance Comparisons of Commercial RT-PCR Systems
6. Restriction Endonucleases Undergo Rigorous QC Testing to Ensure Optimal Performance
7. Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance
8. Versatile Transfection Reagent Offers Low Toxicity and Consistent Performance
9. Stratagenes Human cDNA Microarrays: Perform Gene Expression Profiling and Discover New Genes
10. Challenge the Performance of Your Hot-Start PCRs with FastStart Taq DNA Polymerase and the Novel FastStart High Fidelity PCR System
11. Highly Sensitive microRNA Array Performance
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Perform Microarray Analysis with Limited Bacterial RNA

(Date:3/25/2015)... CAMBRIDGE, Mass. and REYKJAVIK, ... which provides comprehensive capabilities for using the genome to create ... their publication of the largest studies of whole-genome data yet ... in Nature Genetics – are built ... variation, authored by a team of deCODE scientists led by ...
(Date:3/25/2015)... Having added two International locations in Brisbane and ... Global proudly announced recently the organization’s expansion into Poland. ... in the United States, is one of the largest ... real estate. , Saltwood International is the ... advises occupiers of office space, ensuring that its corporate ...
(Date:3/25/2015)... , March 25, 2015 /CNW/ - The Gairdner ... the 2015 Canada Gairdner Awards, recognizing some of the ... Among the world,s most esteemed ... in science and provide a $100,000 (CDN) prize to each ... stronger culture of research and innovation across the ...
(Date:3/25/2015)... , March 25, 2015  CASI Pharmaceuticals, Inc. (Nasdaq: ... acquisition, development and commercialization of innovative therapeutics addressing cancer ... with a commercial focus on China ... trial of its target therapy drug candidate ENMD-2076 in ... Chinese Academy of Medical Sciences in Beijing ...
Breaking Biology Technology:Landmark deCODE Studies Demonstrate the Power of Truly Big Genomics for Improving Medicine Worldwide 2International Organization Expands its Global Reach to Warsaw, Poland 22015 Canada Gairdner Awards Honour Top Medical Discoveries Around The World 22015 Canada Gairdner Awards Honour Top Medical Discoveries Around The World 32015 Canada Gairdner Awards Honour Top Medical Discoveries Around The World 42015 Canada Gairdner Awards Honour Top Medical Discoveries Around The World 52015 Canada Gairdner Awards Honour Top Medical Discoveries Around The World 62015 Canada Gairdner Awards Honour Top Medical Discoveries Around The World 72015 Canada Gairdner Awards Honour Top Medical Discoveries Around The World 8CASI Pharmaceuticals Announces Initiation Of A Phase 2 Trial With ENMD-2076 In Triple-Negative Breast Cancer In China 2CASI Pharmaceuticals Announces Initiation Of A Phase 2 Trial With ENMD-2076 In Triple-Negative Breast Cancer In China 3CASI Pharmaceuticals Announces Initiation Of A Phase 2 Trial With ENMD-2076 In Triple-Negative Breast Cancer In China 4CASI Pharmaceuticals Announces Initiation Of A Phase 2 Trial With ENMD-2076 In Triple-Negative Breast Cancer In China 5
... release is available in Arabic . DUBAI ... possible antidote to high food prices, livestock experts from the ... in Dubai to develop a strategy that eliminates the need ... of Africa, as prevention against the spread of Rift Valley ...
... San Francisco,s leadership in biotechnology has taken another ... partnership to support startup creation and attract industry and ... Mission Bay. "BioSF takes the city,s biotech ... Edwin M. Lee. "This innovative partnership will drive business ...
... LOUIS, June 13, 2011 K-V Pharmaceutical Company (the "Company") ... 10-K for the fiscal year ended March 31, 2011 with ... will be holding an investor conference call today at 4:30 ... provide a general update on other company matters. Participants can ...
Cached Biology Technology:East Africa and Middle East seek plan to keep animal diseases from disrupting livestock trade 2East Africa and Middle East seek plan to keep animal diseases from disrupting livestock trade 3San Francisco and QB3 Launch BioSF, a Program to Enrich the City's Biotech Industry 2K-V Pharmaceutical Company Announces Filing of Fiscal 2011 Form 10-K and Schedules Investor Conference Call 2K-V Pharmaceutical Company Announces Filing of Fiscal 2011 Form 10-K and Schedules Investor Conference Call 3K-V Pharmaceutical Company Announces Filing of Fiscal 2011 Form 10-K and Schedules Investor Conference Call 4K-V Pharmaceutical Company Announces Filing of Fiscal 2011 Form 10-K and Schedules Investor Conference Call 5K-V Pharmaceutical Company Announces Filing of Fiscal 2011 Form 10-K and Schedules Investor Conference Call 6K-V Pharmaceutical Company Announces Filing of Fiscal 2011 Form 10-K and Schedules Investor Conference Call 7
(Date:3/23/2015)... 2015 NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or ... mobile commerce market, announces its biometric payment technology, the Wocket® ... television starting March 30 th . ... New York markets. ... about our new ad campaign following the recent initial shipment ...
(Date:3/20/2015)... 2015 NXT-ID, Inc. (NASDAQ: NXTD ) ... on the growing mobile commerce market, announces its biometric ... the products featured in 2015 "I Want That" International ... EST on the DIY Network.   DIY,s ... , site of the 2015 International CES for ...
(Date:3/18/2015)... SPRINGS, Florida , March 18, 2015 ... well as businesses, new revolutionary smart wallets and apps continue ... pace.  Companies currently entrenched in the mobile payment industry in ... Alibaba Group Holding Ltd. (NYSE: BABA ), Apple ... GOOG ) and Facebook Inc. (NASDAQ: FB ...
Breaking Biology News(10 mins):NXT-ID's Wocket Smart Wallet to Launch New CNBC Regional TV Ad Campaign 2NXT-ID's Wocket Smart Wallet to Launch New CNBC Regional TV Ad Campaign 3NXT-ID's Wocket Biometric Smart Wallet to Air on "I Want That" International CES Special 2NXT-ID's Wocket Biometric Smart Wallet to Air on "I Want That" International CES Special 3Revolutionary Smart Wallet & Mobile Payment Choices Change the Game for Consumers - Company Begins Shipping Newest Digital Wallet to Customers 2Revolutionary Smart Wallet & Mobile Payment Choices Change the Game for Consumers - Company Begins Shipping Newest Digital Wallet to Customers 3Revolutionary Smart Wallet & Mobile Payment Choices Change the Game for Consumers - Company Begins Shipping Newest Digital Wallet to Customers 4Revolutionary Smart Wallet & Mobile Payment Choices Change the Game for Consumers - Company Begins Shipping Newest Digital Wallet to Customers 5Revolutionary Smart Wallet & Mobile Payment Choices Change the Game for Consumers - Company Begins Shipping Newest Digital Wallet to Customers 6
... countries in the wake of the obesity epidemic, and an ... are the most common cause of admission to hospital for ... nerve damage and problems with the blood supply to their ... ulcers which, if they get infected and become gangrenous, can ...
... of University of Helsinki and Sibelius Academy, Helsinki, ... and communication was evaluated using candidate genes associated ... cognitive functions. The data consisted of 343 family ... some professional musicians and/or active amateurs. The musical ...
... difficult to model in mice because their symptoms are ... lethal when transferred to a mouse. The lack of ... diseases. By strategically altering a protein-making molecule, a mouse ... impair feeling and cause paralysis of the arms and ...
Cached Biology News:Protective footwear for diabetics at a high-street price 2Protective footwear for diabetics at a high-street price 3The neurobiology of musicality related to the intrinsic attachment behavior? 2A new mouse model provides insight into genetic neurological disorders 2
UNIVERSAL SCORECARD DNA, 1 EA. * Category: Reagents/Consumables....
mitochondrial proteins (human heart) for 2-D gel electrophoresis *5 mg/mL*...
... GeneMaths XT is undoubtedly the most complete ... advanced concept of layers and subsets makes ... layers for different data outputs, for example ... etc., as well as different subsets of ...
...
Biology Products: