Ovation^® RNA Amplification System V2

What materials are provided with the Ovation RNA Amplification System V2?

The Ovation RNA Amplification System V2 provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and SPIA amplification. The kit also provides nuclease-free water.

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer and a thermal cycler. A UV/Vis spectrophotometer and an Agilent Bioanalyzer will be useful. A real time PCR system may be used for quality control.

What additional consumables are required or useful for the Ovation RNA Amplification System V2?

For the SPIA cDNA purification step, purification columns or SPRI beads are required. Please refer to page 16 of the User Guide for purification options, as well as page 5 for other consumables.

Is the Ovation RNA Amplification System V2 amplification 3’ biased?

Yes. Since the Ovation RNA Amplification System V2 primes the poly(A) tail of transcripts, it is 3’ biased, resulting in coverage up to a range of 1500 bases from the 3’ poly(A) tail.

Has NuGEN performed reproducibility studies on the Ovation RNA Amplification System V2?

Yes. Our studies have included sample-to-sample, lot-to-lot, and operator-to-operator reproducibility.

Can I use the Ovation RNA Amplification System V2 for archiving cDNA?

Yes. Resulting cDNA may be stored at –20°C following purification for at least six months. Ensure the vials are well sealed and avoid multiple freeze/ thaw cycles.

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

• Norgen Biotek Total RNA Purification Kit
• Zymo Research Quick-RNA™ Kits
• Arcturus PicoPure^® RNA Isolation Kit
• Ambion PureLink^® RNA Mini Kit
• Qiagen RNeasy Kits Organic methods such as TRIzol^® Reagent should be subsequently followed with a column-based clean-up method.

Can I use TRIzol^® or other phenol-chloroform based extractions for RNA isolation?

We do not recommend the use of TRIzol^® or similar methods as any carry over of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.

Can I use carrier RNA during RNA isolation?

We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Technical Support for more information.

Do I need to use high quality total RNA?

Yes. Use of lower quality RNA may result in poor performance. One approach to determining RNA quality is the Agilent Bioanalyzer’s RNA Integrity Number (RIN). Clean RNA with a RIN score greater than 7 should amplify well.

How much total RNA input do I need for amplification?

We recommend staying within the specified range of 5 to 100 ng of total RNA starting material. We often suggest 20 ng input as an appropriate starting point. Input greater than 100 ng may adversely affect amplification.

Can I use a total RNA input of less than 5 ng?

The Ovation RNA Amplification System V2 has been validated for total RNA input amounts of 5 to 100 ng. Using input quantities outside the recommended range will affect cDNA yields.

Can I omit quantitation of input RNA?

It is important that RNA input amounts be kept within the 5 to 100 ng range recommended; therefore, we do not recommend omitting quantitation of input RNA.

Can I use mRNA instead of total RNA as starting material?

Purified poly(A)+ RNA has been successfully used as input to the Ovation RNA Amplification System V2. It may be necessary to reduce the input of mRNA to a level comparable to the mRNA present in 5 ng to 100 ng of total RNA.

Can the Ovation RNA Amplification System V2 kits be used for amplification of DNA?

The Ovation RNA Amplification System V2 is designed to amplify mRNA, not DNA. Please contact NuGEN Technical Services for more information on our SPIA-based DNA amplification product offerings.

Can I use the Ovation RNA Amplification System V2 on prokaryotic RNA samples?

The Ovation RNA Amplification System V2 relies on the presence of a poly(A) tail for priming. Therefore, it will not amplify most prokaryotic RNA.

Are there any tissues that will not work with the Ovation RNA Amplification System V2?

We have not encountered any good-quality, clean RNA samples containing poly(A)+ RNA that will not work with the Ovation RNA Amplification System V2.

Does the Ovation RNA Amplification System V2 generate product in a no-RNA reaction?

Due to the high sensitivity inherent in our amplification systems a non-specific product can be generated in the absence of input RNA. However, in the presence of even a very small amount of RNA the amplified cDNA has been demonstrated to be specific. To minimize the potential for carryover of non-specific amplification products into future reactions, we recommend using a low-input (i.e. 50-100 pg) rather than a no-input (i.e. negative) control.

How many rounds of amplification are performed with the Ovation RNA Amplification System V2?

The Ovation Amplification System V2 performs a single round of amplification in fewer than 4 hours. Our products are designed to provide high sensitivity through robust amplification without necessitating a second round of amplification.

Do I need to order specific primers for the amplification?

No. The chimeric DNA/RNA primers provided with the Ovation RNA Amplification System V2 kits are universal. There is no need for additional primers.

Do I have to use the DNA/RNA primers supplied with the kit?

Yes. The Ovation RNA Amplification System V2 was designed and optimized to work with the primers provided. The use of other primers with the Ovation RNA Amplification System V2 is not supported.

Why is the purification of cDNA step optional?

cDNA purification is required for microarray applications. cDNA for use in with an Encore labeling module or other supported microarray labeling protocol must be purified to allow for accurate quantitation of cDNA as well as ensure proper performance of the labeling method. If the amplified cDNA is to be used solely for qPCR analysis alone, then purification is optional. Purification, however, will enable mass normalization of cDNA input into the qPCR reaction, potentially reducing variability.

What are the recommended storage conditions for amplified cDNA?

The amplified cDNA may be stored at –20°C. Ensure the vials are well sealed and avoid multiple freeze thaw cycles.

Where can I safely stop in the protocol?

You may safely stop after the SPIA Amplification step or final SPIA cDNA Purification and store the cDNA at -20°C.

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP beads in our kits and observe no difference in performance between products.

What magnetic separation devices do you recommend for the SPRI bead purifications?

Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:

• Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
• Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
• Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN workflow. This is also helpful to gain familiarity with the purification workflow.

How can I ensure maximum recovery of sample from the SPRI bead purification?

• Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
• Ensure that the beads are fully resuspended in solution before adding to the sample.
• Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
• Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

What is the amplification efficiency of the Ovation RNA Amplification System V2?

Based on qPCR results of a collection of housekeeping genes, amplification efficiency ranges from 1,000- to 10,000-fold or higher depending on the input amount.

How can I ensure good yields at the cDNA purification step?

In order to maximize yields, we recommend the following:

• Do NOT use cold water for the elution step. Use the D1 Nuclease-free Water included in the Ovation RNA Amplification System V2 kit at room temperature.
• Do NOT spin the columns at an incorrect speed. Strictly adhere to the guidelines in the user guide.
• Use a fresh dilution of ethanol from a fresh stock for any washing steps.
• Vortex the eluted cDNA sample prior to measuring the absorbance.

How much cDNA yield can I expect from one reaction of amplification?

For a standard reaction the expected yield is 4–7 μg of amplified cDNA.

Is the cDNA yield dependent upon the quantity of input total RNA?

The total yield of cDNA is not directly dependent upon input RNA amount due to upper limit constraints on cDNA production in the reaction.

What is the size range of cDNA generated by the Ovation RNA Amplification System V2?

As measured with an Agilent Bioanalyzer, the majority of amplified SPIA cDNA is between 200 bases and 2 Kb in length.

Should I purify the cDNA before determining the concentration?

Yes. The primers and reagents present in the amplified cDNA will interfere with accurate quantitation. Details on measuring the concentration of cDNA are included in the User Guide.

What types of arrays work with the cDNA generated with the Ovation RNA Amplification System V2?

The cDNA generated with the Ovation RNA Amplification System V2 has been validated on Affymetrix GeneChip arrays, Agilent Dual-Mode Gene Expression Arrays and Illumina Whole Genome Expression BeadChips when used in conjunction with the appropriate Encore labeling module or protocol. Contact NuGEN Technical Services for more information.

For quantitative real time PCR applications, what is the optimal distance from the 3´ poly(A) tail for design of primer probe sets?

Due to the 3’ oriented amplification mechanism of the Ovation RNA Amplification System V2, we recommend primer/probe sets to be designed within the first 1.5 Kb from the poly(A) tail.