Navigation Links
Synthesize High Yields of Biotinylated aRNA

MessageAmp II-Biotin Enhanced: A Single Round aRNA Amplification Kit
Ambion now offers an improved MessageAmp II Kit with biotin-11-UTP. MessageAmp II-Biotin Enhanced provides a complete single round amplification kit for biotin array analysis. The biotin modified UTP and ratio of labeled to unlabeled UTP were selected based on superior performance and aRNA yield. The biotinylated nucleotide is supplied as a pre-mixed NTP at an optimized ratio, providing convenient, consistent labeling.

Highest amplification in a single roundup to 10,000-fold for most samples

Enough aRNA for Affymetrix GeneChip analysis from just 50 ng total RNA

Optimized NTP mix containing biotinylated UTP reduces set-up time and maximizes labeling

Reduce costs by 45%

T7 RNA Polymerase Linear Amplification is the Gold Standard

Microarrays measure the expression levels of thousands of genes. One limitation of this technology, however, is the need for large amounts of labeled target RNA for hybridization to the microarray. This limitation is overcome by using a T7 linear amplification protocol commonly referred to as the Eberwine Method. This method most accurately maintains the initial mRNA expression profile and has been the gold standard for microarray target preparation [1]. Biotin, incorporated as biotinylated nucleotide tri phosphates during the in vitro transcription (IVT) step, is the preferred label for most commercial microarray platforms. The highly specific and efficient binding of streptavidin-dye conjugates (phycoerythrin,
Cy dyes) to biotinylated RNA/oligonucleotide probe hybrids is the basis of highly sensitive fluorescence detection. Ambion has incorporated biotin labeling reagents into the MessageAmp II Kit to provide a complete microarray aRNA preparation kit, the MessageAmp II-Biotin Enhanced Kit.

Higher Yields of Labeled aRNA; Only 1 Round of Amplification

Both biotin incorporation and further optimization of the IVT reaction has resulted in increased sensitivity of microarray transcript detection. The increased sensitivity allows the use of less input RNA-50 ng of input RNA in a single round of amplification provides enough aRNA for array analysis. Amplified RNA yields derived from nine different tissue and cell sources and four different input amounts are shown in Figure 1. While length of the aRNA synthesized will largely depend on RNA sample quality, the cDNA and IVT reaction conditions in this kit produce longer biotinylated aRNA compared to the market leader. Figure 2 shows comparable Agilent bioanalyzer scan results from labeled aRNA produced with MessageAmp II-Biotin Enhanced Kit and unlabeled aRNA produced with MessageAmp II Kit. The resulting improvements also give the MessageAmp II-Biotin Enhanced Kit a higher number of Present calls than the market leader (Figure 3).


Figure 1. aRNA Yield from Nine Different Sources. Four different amounts of total RNA from nine different sample types were amplified using the MessageAmp II-Biotin Enhanced Kit. Average aRNA yields from triplicate reactions are shown for 4 or 14 hr in vitro transcription (IVT) reactions. These data are useful for determining both the amount of total RNA needed to obtain enough labeled aRNA for array hybridization (typically ~10 g) and the optimal length of IVT incubation. Note that there is a ~3-fold difference in aRNA yield between some samples. With most RNA sources, 50100 ng of input total RNA amplified with the MessageAmp II-Biotin Enhanced Kit using a 14 hr IVT incubation will yield enough labeled aRNA for microarray hybridization.

Figure 2. Electropherogram of Biotin-labeled vs. Unlabeled aRNA. Identical HeLa cell total RNA samples (1000 ng) were amplified with either the MessageAmp II-Biotin Enhanced Kit to produce biotin-labeled aRNA or the MessageAmp II Kit to produce unlabeled aRNA. The IVT reactions were carried out for 4 hr. Equivalent mass amounts from each reaction were analyzed on an Agilent bioanalyzer. The shift in aRNA size from the MessageAmp II-Biotin Enhanced sample results from the incorporation of biotin into the aRNA.

Figure 3. Number of Present Calls Detected on Affymetrix Human Genome Focus Arrays. The indicated amounts of total RNA from HeLa cells was amplified in triplicate using either the Mess ageAmp II-Biotin Enhanced Kit (MA II-Biotin) or another manufacturers aRNA labeling kit (Kit A). Two IVT incubation times (4 and 14 hrs) were tested with 1000 ng of total RNA input. Amplification of as little as 50 ng total RNA with the MessageAmp II-Biotin Enhanced Kit produced a comparable number of Present calls as 1000 ng input RNA that was amplified with Kit A. Furthermore, the amplification reaction can be completed in one day.

Hybridization of the biotinylated aRNA produced from the MessageAmp II-Biotin Enhanced Kit to Affymetrix GeneChip Arrays was used to assess the sensitivity and reproducibility of the method and reagents. Increased sensitivity can be measured several ways. One indication of sensitivity is the number of Present calls calculated by the GeneChip Operating Software (Figure 3). Additional analysis of the distribution of signal intensities indicates that the new MessageAmp II-Biotin Enhanced Kit produces a higher mean value. This means that a there was a higher signal intensity across the array (Figure 4). This results in more reproducible array results and increased sensitivity of lower expressed transcripts.

Figure 4. Higher Signal Intensity Distribution on Affymetrix Human Genome Focus Arrays. Distributions of log2 signal intensity values were plotted for the MessageAmp II-Biotin Enhanced Kit and another manufacturers amplification kit (Kit A). The median values for each of the MessageAmp II-Biotin Enhanced plots were similar for total RNA inputs ranging from 50 to 1000 ng. In contrast, signal intensities produced by Kit A at the 1000 ng total RNA input level was markedly reduced. The box and whiskers plot ab ove each histogram indicate the median value (vertical center line), interquartile range (left and right sides of box), and the 10% and 90% quantiles (ends of line). Each plotted distribution represents the average log2 signal intensity of triplicate target preparations and hybridizations. IVT reactions were carried out for 4 or 14 hrs as indicated. The data were normalized within each group using Robust Multichip Average (RMA) (See sidebar, Data Analysis: Robust Multichip Average (RMA) and Affymetrix Algorithms).

Biotin Incorporation

Biotin-11-UTP serves as the label in the MessageAmp II-Biotin Enhanced Kit. Only a single biotinylated NTP is used because studies at Ambion and elsewhere [2] showed that in previous dual labeling experiments using biotin-UTP and biotin-CTP, the signal contributed by biotin-CTP was minimal, and relative signal intensity between genes was not altered with the use of a single biotin UTP label.

While various linker arm sizes (e.g., Biotin-11-UTP, Biotin-16-UTP) did not affect modified UTP incorporation into aRNA during IVT, the longer linker arms slightly impeded subsequent purification of the aRNA. Biotin-11-UTP provided the highest yields of aRNA.

The ratio of biotin UTP to unlabeled UTP was optimized to provide the highest yield of aRNA and the highest signal:noise on Affymetrix GeneChip Arrays. A fairly wide range of concentrations of Biotin-UTP (25 60%) can be used for acceptable detection, whereas at lower concentrations the signal drops dramatically, and at higher concentrations both the noise increases and the aRNA yield decreases.

Ambion realizes that microarray users may prefer to continue using their own biotin-labeling method, or to experiment with different labeling conditions. To accommodate this we offer two Biotin-UTP nucleotides (Biotin-11-UTP and Biotin-16-UTP) separately, which can be used with the standard MessageAmp II Kit.

MessageAmp II-Biotin Enhanced Kit Format

The MessageAmp II-Biotin Enhanced Kit provides enough reagents for 20 single round amplification reactions. It can be used in combination with the MessageAmp II Kit for small samples that require 2 rounds of amplification (MessageAmp II for the first round and MessageAmp II-Biotin Enhanced in the second round for labeling). Biotin-UTP is provided premixed with the other rNTPS for convenience and consistent, optimized labeling.

Scientific Contributors

Robert Setterquist, Mike Wilson, Charles Johnson, Shika Agarwal, Sharmili Moturi Ambion, Inc.

back to top

Ordering Information
Cat# Product Name Size 1334 MEGAscript T7 Kit 40 rxns 1751 MessageAmp II aRNA Amplification Kit 20 rxns 1791 MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit 20 rxns 8450 10 mM Bio-11-UTP 25 l (250 nmol) 8451 75 mM Bio-11-UTP 30 l (2250 nmol) 8452 10 mM Bio-16-UTP 25 l (250 nmol) 8453 75 mM Bio-16-UTP 30 l (2250 nmol)


Page: All 1 2 3 4 5 6 7

Related biology technology :

1. Rapidly Synthesize Large Amounts of Capped RNA
2. Yeast Protein Production System Features High Yields and One-Step Purification
3. ArrayScript RT: Maximize Yields of Full-length cDNA
4. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
5. Translate High Yields of Active Protein
6. Translate High Yields of Biologically Active Proteins
Post Your Comments:

(Date:10/8/2015)...  The ALS Association, in partnership with Prize4Life, is ... revolutionize communication technology solutions for people living with ALS. ... (amyotrophic lateral sclerosis) is a progressive neurodegenerative disease that ... spinal cord. Eventually, people with ALS lose the ability ... to total paralysis and death within two to five ...
(Date:10/8/2015)... ... ... Talon Innovations, a provider of Precision Machined Products and Assemblies ... one of three finalists for the Minnesota Business magazine’s Manufacturing Award in the category ... was recognized as a nominee for this competitive award. , The “Best in ...
(Date:10/7/2015)... , Oct. 7, 2015 Boston ... the start of the Boston Scientific Connected Patient ... advancements in the use of remote patient monitoring to ... designers and entrepreneurs can submit their ideas and collaborate ... ( ). Cambridge, MA ...
(Date:10/7/2015)... -- ContraVir Pharmaceuticals, Inc. (Nasdaq: CTRV ) ("ContraVir" or ... and commercialization of targeted antiviral therapies, today announced the ... of its common stock and warrants to purchase up ... a fixed combined price to the public of $3.00. ... issued separately.  The warrants will be exercisable for a ...
Breaking Biology Technology:
... , , HUNTSVILLE, Ala., ... in nucleic acid therapeutics and delivery, announced today that Jason ... Pharmacology to Vice-President of Preclinical Research and Development. , ... management and scientific leadership experience in the biotechnology industry and ...
... , , SAN MARINO, Calif., ... has formed a wholly owned subsidiary called MetaCytoLytics, Inc. This ... of Colorado Professor M. Karen Newell called "metabolic disruption technology" ... generate energy from glucose or from fatty acids. That ...
... , , PASADENA, Calif., Aug. 6 AutoImmune ... $86,000, or $0.01 per share basic and diluted, for the three ... $86,000, or $0.01 per share basic and diluted, for the three ... 30, 2009, the net loss was $256,000, or $0.02 per share ...
Cached Biology Technology:
(Date:9/10/2015)... 10, 2015 Report Details ... Selling Opportunities and Revenue Prospects to Help You ... biologics, especially new drug classes? Get the latest ... with exclusive market data and industry knowledge, benefitting ... results, opportunities and sales predictions. Visiongain,s new ...
(Date:9/9/2015)... , Sept. 9, 2015  NuData Security ... and solution-based milestones, furthering the company,s commitment to ... online fraud. NuData Security,s online fraud ... the company,s growth cycle. The product combines continual ... determine good user behavior from fraudulent behavior, allowing ...
(Date:9/2/2015)... 2015 Security of data and assets ... government organizations, telecommunications, hospitals, as well as individuals. ... being used to prevent unauthorised access. Technological advancements ... individuals could gain access to secure premises after ... hardware devices and numeric codes. However, hackers also ...
Breaking Biology News(10 mins):
... for Medical Research have glimpsed two proteins working together ... nucleus and its exterior compartment, the cytoplasm. The research ... is found in organisms from yeast to humans does ... Investigator Sue Jaspersen, Ph.D., focused on a protein called ...
... at high elevations found in residents of the Tibetan ... related to contemporary Sherpa. These genes were passed on ... mixing, and then amplified by natural selection in the ... by scientists from the University of Chicago and Case ...
... that electrically stimulate the auditory nerve have granted ... people worldwide who otherwise would be totally deaf. Existing ... transmitter about an inch in diameter be affixed to ... joint microphone and power source that looks like an ...
Cached Biology News:
... CLS number is a new ... match Cornings product number. If ... order under the old Sigma-Aldrich ... service for assistance. ID clarifier: ...
... The GenElute Gel Extraction Kit combines silica- binding ... vacuum column format. DNA fragments of interest are ... solubilizing the gel. The Gel Solubilization Solution can ... in either TBE or TAE buffer. Extracted DNA ...
... are designed for the rapid purification of linear ... gels. The DNA band is excised from an,agarose ... within the base of the column are a ... and impurities back while allowing DNA to selectively ...
... Ultra Agarose Spin Kit is an advanced system for ... purified DNA can be used directly for any downstream ... DNA is ready to use in 56 minutes ... used for TAE and TBE gels No specialised ...
Biology Products: