Estibaliz Aldecoa-otalora Astarloa is the primary scientist performing RNA amplification experiments at the Genomics Centre at Kings College, London. “Our testing of the MessageAmp Premier Kit has led us to integrate it into our protocol for amplification of total RNA samples for our microarray studies. The kit reduces the amount of starting total RNA needed, allowing us to perform microarray studies on a wider range of samples, including clinical samples, and still save a portion of the sample for subsequent validation studies.” —Estibaliz Aldecoa-otalora Astarloa

The Microarray Analysis Facility first turned to the Ambion MessageAmp RNA Amplification product line to address the smaller samples researchers were providing. The MessageAmp RNA Amplification Kits yielded sufficient labeled RNA from these limited samples for Affymetrix GeneChip hybridization which typically requires 15-20 μg labeled cRNA per GeneChip. Using their prior protocol, at least 5 µg total RNA sample was needed for labeling and subsequent hybridization. By contrast, only 100 ng of total RNA was needed with the Ambion MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit.

Recently the Microarray Analysis Facility has migrated to the new Ambion MessageAmp Premier RNA Amplification Kit. This kit substantially streamlines the RNA amplification and labeling process used in previous MessageAmp products and competing protocols. Improvements to the new kit include: 1) single tube reactions that include reverse transcription, in vitro transcription, and RNA amplification steps; 2) shorter incubation time; 3) Master Mix formulation to reduce pipetting and handling steps; and 4) use of magnetic bead based purification to provide higher yields with more consistent results. These modifications result in a protocol that can use as little as 20 ng total RNA to product enough aRNA for Affymetrix GeneChip analysis when an overnight in vitro transcription reaction is used. And it is possible to start with 100 ng total RNA, and with a 4 hr in vitro transcription reaction, hybridize to a GeneChip in a single day.

Estibaliz, the primary scientist performing the RNA amplification procedures, commented, “I like the new kit—the procedure is shorter, the yields are higher…and I especially like the use of magnetic beads for purification as they make it fast and easy to do.”

Prior to adopting the Ambion MessageAmp Premier RNA Amplification Kit for processing the core facility’s samples, Estibaliz and colleagues performed a validation experiment to test the new Ambion MessageAmp Premier product for yield and correlation with the MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit that they had been using. 100 and 20 ng of each of two RNA samples—a single pooled test sample provided by a customer, and a positive control from human cell line A431—were amplified for 14 hr using the protocol provided for each kit, and resulted in the amplified RNA (aRNA) yields shown in Figure 1.

aRNA generated by amplification of 100 ng of the test sample by both kits was hybridized to Affymetrix Mouse430A_2 Chips. The data from these two samples was highly correlated (R2=0.99; Figure 2).

	Sample Type Total RNA Input Kit used for Amplification aRNA Yield Test Sample 100 ng MessageAmp II 14.65 µg A431 100 ng MessageAmp II 167.13 µg Test Sample 100 ng MessageAmp Premier 26.72 µg Test Sample 20 ng MessageAmp Premier 8.14 µg A431 100 ng MessageAmp Premier 60.75 µg A431 20 ng MessageAmp Premier 17.61 µg
	Figure 1. aRNA Yields using MessageAmp™ aRNA Amplification Kits. 100 and 20 ng of each of two RNA samples—a single pooled test sample provided by a customer, and a positive control from human cell line, A431 (Applied Biosystems) —were amplified for 14 hr using the protocol provided for the MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit and the MessageAmp Premier RNA Amplification Kit, respectively. The resulting aRNA yields are shown.
		Figure 2. Ambion® MessageAmp™ Premier RNA Amplification Kit Microarray Data Highly Correlated with MessageAmp II-Biotin Enhanced Single Round aRNA Amplification Kit Data. Replicates of a single pooled total RNA test sample (100 ng) provided by a customer were amplified for 14 hr by each of the MessageAmp Kits using the respective kit protocols. Resulting aRNA was hybridized to Affymetrix® Mouse430A_2 GeneChips® and correlation analysis was performed. The data from these two samples was highly correlated (R2=0.99).

Scientists seeking to increase the throughput of their real-time PCR workflows and deliver results faster are quickly adopting “fast” real-time PCR. Fast real-time PCR allows researchers to conduct real-time PCR experiments in less time by utilizing an integrated system of optimized instruments and specialized chemistries that enable faster reaction times without compromising data quality. Dr. Dirk Hincha and his colleagues at the Max-Planck Institute of Molecular Plant Physiology in Germany are successfully using the Fast SYBR® Green Master Mix as part of a fast real-time PCR workflow to study different aspects of plant stress tolerance.

Dr. Dirk K. Hincha leads the Transcript Profiling Group at the Max-Planck Institute of Molecular Plant Physiology in Potsdam, Germany. He completed a Ph.D. in Plant Sciences at the University of Würzburg, Germany and worked subsequently at the University of California, Davis and the Free University of Berlin, Germany, before joining the Max-Planck Institute.   "Fast SYBR® Green Master Mix cuts measurement time in half without compromising data quality and reliability. This makes large scale projects more feasible.” —Dr. Dirk K. Hincha Fast SYBR® Green Master Mix Fast SYBR Green Master Mix delivers highly sensitive and reproducible real-time PCR results in less than half the time when used with Applied Biosystems fast-enabled, real-time PCR instruments. The Fast Master Mix also offers high specificity by employing a new highly purified AmpliTaq® Fast DNA Polymerase, UP, in an optimized formulation to minimize non-specific PCR products. SYBR Green dye is a cost-effective and easy to use nucleic acid labeling method for real-time PCR which is commonly used in applications such as gene expression and microarray validation.

Scientists seeking to increase the throughput of their real-time PCR workflows and deliver results faster are quickly adopting “fast” real-time PCR. Fast real-time PCR allows researchers to conduct real-time PCR experiments in less time by utilizing an integrated system of optimized instruments and specialized chemistries that enable faster reaction times without compromising data quality. Dr. Dirk Hincha and his colleagues at the Max-Planck Institute of Molecular Plant Physiology in Germany are successfully using the Fast SYBR® Green Master Mix as part of a fast real-time PCR workflow to study different aspects of plant stress tolerance.

Plant Profiling at the Max-Planck Institute
Dr. Hincha leads the Transcript Profiling Group at the Max-Planck Institute of Molecular Plant Physiology. The research group studies the dynamics of plant metabolism in the context of the plant system as a whole. Since the system is more than a collection of genes and gene products, the research focus is on how these components dynamically interact over time and under different conditions. By combining traditional biological approaches with techniques relevant to functional genomics, they are forming a holistic view of structure, function, dynamics and regulation of entire plant genomes, proteomes and metabolomes. Dr. Hincha and his colleagues are working on different aspects of plant stress tolerance, i.e. tolerance to freezing, drought and desiccation. They use approaches ranging from classical physiology through biochemistry to metabolomics and transcriptomics.

Speed Without Compromise
The Fast SYBR Green Master Mix provides scientists with a new choice in fast-enabled PCR chemistries that delivers results in less than half the time of standard SYBR green reagents. Dr. Hincha used the Power SYBR Green Master Mix in his research, and has recently tried the Fast SYBR Green Master Mix. He commented, “The Fast SYBR Green Master Mix cuts measurement time in half without compromising data quality and reliability. This makes large scale projects more feasible. The higher throughput is a huge benefit.”

Accelerated Real-Time PCR Workflow
Dr. Hincha currently uses one StepOnePlus™ and four 7900HT Fast Real-Time PCR Systems in his laboratory. The Fast SYBR Green Master Mix combined with these fast-enabled, real-time PCR instruments has accelerated his workflow. Dr. Hincha says, “We often have projects where we need to run up to 100,000 measurements. Whereas time constraints limit the possible duration of large-scale experiments, reducing measurement time through use of Fast SYBR Green Master Mix makes such projects more feasible, where time constraints limit the possible duration of large-scale experiments.”

NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit

The rapid and convenient NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Kit assay provides a monitoring system for influenza resistance to viral inhibitors for research purposes and can supply highly sensitive viral quantitation measurements. While at the CDC, the Australian virologist, Dr. Bruce Mungall, evaluated several common sample storage and handling procedures for their influence on the sensitivity and reproducibility of this assay.

Based at CSIRO’s Australian Animal Health Laboratory (AAHL) in Geelong (outside of Melbourne), Dr Bruce Mungall is a research scientist with expertise in developing and evaluating antivirals. Mungall joined CSIRO in 2004 following postdoctoral positions at Emory University School of Medicine and the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, USA. Working in the Strain Surveillance Section of the Influenza Branch at CDC, Dr Mungall established assays to routinely assess the development of resistance to the new class of anti-influenza drugs, the neuraminidase inhibitors. In addition, his research has included the development of therapeutics effective against two recently emerged zoonotic viruses: Hendra virus and Nipah virus. “The kit is performing exactly as expected and will certainly fulfill our requirements in this area.” —Dr. Bruce Mungall

Quantitating Neuraminidase Activity as a Measure of Viral Drug Resistance
Neuraminidase (NA) is a surface component of influenza virus that facilitates release of virus from cells via cleavage of sialic acid residues from glycoproteins on the surface of infected cells. NA inhibitor drugs are the primary anti-influenza therapeutics that will be relied upon to contain a potential influenza outbreak. Thus, global monitoring of influenza strains for resistance to viral inhibitors is essential for studying epidemiology of viral strains and their mutations, and for understanding the efficacy of antiviral therapeutics in the event of a significant influenza outbreak.

NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit
In consultation with worldwide public health protection agencies, including the Centers for Disease Control (CDC) and member laboratories of the Neuraminidase Inhibitor Susceptibility Network (NISN), Applied Biosystems has integrated its 1,2-dioxetane chemiluminescent technology into a complete detection kit for measuring the level of NA inhibitor resistance in influenza virus isolates [1–4]. The resulting NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit provides a highly sensitive, rapid, and standardized detection assay for quantitating the level of NA inhibitor resistance of virus isolates from avian, equine, human (types A and B), and porcine sources.

Figure 1. Effect of Long-term Storage at 4°C on Neuraminidase Activity of Virus Stocks. Regression analysis of NA activity (signal-to-noise value; S/N) for virus stocks prepared freshly (left panel), stored 4 months at 4°C (center panel), or stored 6–8 months (right panel) at 4°C, versus matched stocks stored at –70°C.

Chemiluminescent NA-Star Assay for Neuraminidase Activity Quantitation
The NA-Star neuraminidase assay is based on specific hydrolysis of the chemiluminescent NA-Star substrate by neuraminidase. The assay is very convenient and rapid compared to culture-based methods, hemagglutination assays, or immunoassay methods for virus quantitation. If a correlation between NA-Star assay results and other titering methods is established for particular virus strains, the NA-Star assay has the potential to provide highly sensitive viral neuraminidase quantitation measurements.

Effects of Storage and Handling on Neuraminidase Activity
As with any enzymatic reaction, sample storage, the number of freeze-thaw cycles, and the addition of chemicals (particularly detergents) can all profoundly influence NA activity of virus samples. Dr. Bruce Mungall addressed the impact of these factors on the sensitivity and reproducibility of the NA-Star NA activity assay. NA assays were performed by diluting the virus stock in assay buffer, omitting addition of NA inhibitor, and incubating with diluted NA-Star Substrate for 10 min at room temperature. Accelerator solution was added using the injector device associated with the luminometer (the recommended method) and light emission was read immediately.

Effect of Virus Storage Conditions and Freeze-thaw Cycles
The CDC Influenza Laboratories routinely store bulk virus stocks at 4°C and aliquots at –70°C. This provided an opportunity to compare NA activity in virus stocks stored for different periods of time at 4°C to determine if storage conditions would bias observed NA activities. 125 paired virus samples were selected and grouped according to length of storage at 4°C: freshly prepared; approximately 4 months; and between 6 and 8 months (Figure 1). These groups were then matched with –70°C stocks that had not been thawed since storage.

NA activity was determined for each virus stock (1:5 dilution). Regression analysis determined the degree of correlation between values obtained for each sample (Figure 1). In general, the correlation of NA activity between stocks stored at 4°C and –70°C decreased as the length of time stored at 4°C increased, indicating that NA activity declines during storage at 4°C.

To evaluate the effect of freeze-thaw cycles on virus NA activity, aliquots of master stocks of three different virus strains were subjected to ≤6 freeze-thaw cycles. As much as 50% of the total NA activity can be lost with as little as two freeze thaw cycles (not shown).

The results of both the 4°C storage and the freeze-thaw experiments indicate that storage artifacts must be strictly managed to enable an accurate determination of viral load in unknown samples. It is also recommended that aliquots of master stocks be prepared for each virus standard such that aliquots are used only once in an assay.

Effect of Detergents
Detergents are commonly used to solubilize virus during cell and tissue recovery procedures. To evaluate the direct effect of detergents on NA activity, 16 different virus stocks were assayed in the absence and presence of either 0.1% Triton® X-100, a commonly used nonionic detergent, or 0.1% deoxycholate, a biological detergent used to lyse cells and solubilize cellular and membrane components. Both detergents increased observed NA activity for all viruses, except for H1N1 subtypes in the presence of Triton X-100 (Figure 2). The observed increase in NA activity is most likely due to solubilization of individual NA molecules from viral particles, making them more accessible for substrate interactions.

	Figure 2. Effect of Detergents on NA Activity of Virus Stocks. Virus stocks of the indicated types (6 type B viruses, 2 type H1N1 and 8 type H3N2 viruses) were assayed for NA activity in the absence or presence of either 0.1% Triton® X-100 or 0.1% deoxycholate (DoCh). Mean ± S.E. for each group of virus types is shown. S/N: Signal-to-noise ratio; S/N was calculated as the ratio of the signal for each dilution to that of the negative control samples [either culture media from uninfected cells (recommended) or assay buffer alone.

This work highlights the need for careful sample preparation and handling to ensure that differences in neuraminidase activity observed in either viral NA quantitation assays or NA inhibition assays reflect differences in the amount of NA activity present or its differential sensitivity to inhibitors.

References
1. Mungall BA, Xu X, and Klimov A. (2003) Assaying susceptibility of avian and other influenza A viruses to Zanamivir: Comparisons of fluorescent and chemiluminescent neuraminidase assays. Avian Diseases 47:1141–1144.
2. Wetherall, NT, Trivedi T, Zeller J, Hodges-Savola C, McKimm-Breschkin JL, Zambon M, and Hayden FG. (2003) Evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: Report of the Neuraminidase Inhibitor Susceptibility Network. J Clin Microbiol 41(2):742–750.
3. Mungall BA, Xu X, and Klimov A. (2004) Surveillance of influenza isolates for susceptibility to neuraminidase inhibitors during the 2000-2002 infl uenza seasons. Virus Research 103:195–197.
4. Monto AS, McKimm-Breschkin JL, Macken C, Hampson AW, Hay A, Klimov A, Tashiro M, Webster RG, Aymard M, Hayden FG, and Zambon M. (2006) Detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the fi rst 3 years of their use. Antimicrobial Agents and Chemotherapy 50(7):2395–2402.

NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Kits are For Research Use Only. Not for use in diagnostic applications.

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