Frequently Asked Questions
Ovation™ Whole Blood
• Ovation™ RNA Amplification System V2
(Cat # 3100-12, 3100-60)
• Ovation™ WB Reagent
(Cat # 1300-12, 1300-60)
• FL-Ovation™ cDNA Biotin Module V2
(Cat # 4200-12, 4200-60)
Q1. What NuGEN products are required for the Ovation™ Whole Blood Solution?
There are 3 NuGEN products that comprise the Ovation™ Whole Blood Solution, all are required for target preparation from whole blood RNA for GeneChip® array analysis:
- The Ovation™ RNA Amplification System V2 (Cat.# 3100); RNA amplification module
- The Ovation™ WB Reagent (Cat.# 1300); an optimized reagent for whole blood RNA amplification
- The FL-Ovation™ cDNA Biotin Module V2 (Cat.# 4200); a fragmentation and labeling module
Q2. 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 and a real time PCR system may be used for quality control.
Q3. What additional consumables are required for the use of Ovation™ RNA Amplification System V2?
Zymo Research DNA Clean and Concentrator™-25 columns (Zymo Research, Cat. #D4005) is required for purification of amplified cDNA.
Q4. 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 of greater than 7 should amplify well.
Q5. How much total RNA input do I need for amplification?
We recommend staying within a range of 20 to 50 ng of whole blood total RNA. Greater RNA input may adversely affect amplification, while lower input may affect yields or quality of amplified cDNA.
Q6. What is the dynamic range of input mRNA copies that are linearly amplified?
Our studies demonstrate linear amplification of transcripts present at 100 to 100,000,000 copies in a 5 ng sample of HeLa RNA. Different input amounts of RNA from different tissues such as whole blood may affect the lower limit of detection.
Q7. Can I use a total RNA input of less than 20 ng?
The Ovation™ RNA Amplification System V2 has been validated for total RNA input amounts of 5 to 100 ng, however for whole blood amplifications we strongly recommend staying within the 20-50 ng range. Using lower input amounts will result in lower performance.
Q8. Can I omit quantitation of input RNA?
No, we do not recommend omitting quantitation of input whole blood RNA.
Q9. Can I use mRNA instead of total RNA as starting material?
Purified poly(A) RNA has been successfully used as input to the Ovation™ System. It will be necessary to use much lower amounts of input mRNA, comparable to mRNA present in 5-100 ng of a total RNA sample from the same tissue.
Q10. Is the Ovation™ System amplification 3 prime biased?
Since the Ovation™ RNA Amplification System V2 primes the poly-A tail of transcripts, it is 3 prime biased, resulting in coverage up to a range of 1500 bases from the 3’ poly A tail.
Q11. How much cDNA yield can I expect from one reaction of Ovation™ RNA Amplification System V2 used with the Ovation™ WB Reagent?
For a standard reaction the expected yield is 5-12 μg of amplified cDNA.
Q12. 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.
Q13. What is the amplification efficiency of the Ovation™ System?
Based on qPCR results of a collection of housekeeping genes tested on HeLa RNA, amplification efficiency ranges from 1,000 to 10,000 fold or higher depending on the input amount.
Q14. What is the size range of cDNA generated by the Ovation™ System?
As measured with an Agilent Bioanalyzer, the majority of amplified SPIA™ cDNA is between 200 bases and 2000 bases in length. After fragmentation, 80% of product falls below 200 bases with an average peak at 85 bases.
Q15. Has NuGEN performed reproducibility studies on the Ovation™ System?
Our studies have included sample to sample, lot to lot, and operator to operator reproducibility.
Q16. Can DNA be used as input for the Ovation™ System?
The Ovation system is designed to amplify mRNA.
Q17. Can I use the Ovation™ System on prokaryotic RNA samples?
The Ovation™ System relies on the presence of a poly-A tail for priming. Therefore, it will not amplify most prokaryotic RNA.
Q18. Are there any tissues that will not work with the Ovation™ System?
We have not encountered any good quality, clean RNA samples containing poly(A) RNA that will not work with the Ovation™ System.
Q19. Does the Ovation™ System generate product in a no-RNA reaction?
As with most amplification systems, non-specific product is generated using the Ovation™ System in the absence of input template. Array and qPCR analysis show these amplification products to be non-specific.
Q20. How many rounds of amplification are performed with the Ovation™ System?
The Ovation™ System performs a single round of amplification in less than 4 hours. Our products are designed to provide high sensitivity through robust amplification without necessitating a second round of amplification.
Q21. Do I need to order specific primers for the amplification?
The chimeric DNA/RNA primers provided with the Ovation™ System kits are universal, and there is no need for additional primers.
Q22. Do I have to use your DNA/RNA primers?
The Ovation™ System was designed and optimized to work with the primers provided. The use of other primers with the Ovation™ System is not supported.
Q23. Do I need to change the sense of the cDNA for use on oligo arrays?
The Ovation™ RNA Amplification System generates antisense cDNA. There is no need to change the sense.
Q24. Can I fragment the cDNA?
Yes, the amplified cDNA fragmentation and labeling can be done using the FL-Ovation™ cDNA Biotin Module V2.
Q25. Can I use the Ovation™ RNA Amplification System V2 for archiving cDNA, and what are the recommend storage conditions?
cDNA may be stored at -20°C following purification for at least six months. Longer term stability tests are in progress. Ensure the vials are well sealed and avoid multiple freeze thaw cycles.
Q26. Are there any excerptions to the protocol when using the amplified cDNA from this procedure with the FL-Ovation™ cDNA Biotin Module V2?
We recommend following the procedure as outlined in the FL-Ovation™ cDNA Biotin Module V2, the only exception for whole blood RNA is that the cDNA input to each F&L reaction is 4.4 μg.
Q27. For quantitative real time PCR applications, what is the optimal distance from the 3 prime poly(A) tail for design of primer probe sets?
Due to the amplification mechanism of the Ovation™ System, we recommend primer/probe sets to be designed within the first 1,500 bases from the poly-A tail.
Q28. What are the Ovation™ RNA Amplification System V2 incubation temperatures?
First strand primer annealing = 65°C
First strand synthesis = 48°C
Second strand synthesis = 37°C
SPIA™ amplification = 48°C (with a pause step)
Fragmentation and Labeling = 50°C
Q29. Where can I safely stop in the Ovation™ RNA Amplification System V2 protocol?
You may safely stop after cDNA amplification or after the purification and store the cDNA at -20°C. The fragmented and labeled cDNA may also be stored at -20°C or short term storage or at -80°C for longer term storage.
Q30. 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™ System 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 O.D.
Q32. 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 this user guide.
Q33. Do you recommend any specific whole blood RNA isolation methods?
We do not specifically require one method of RNA preparation, however for whole blood samples the Qiagen PAXgene products for stabilization and RNA isolation have been validated and are compatible with the Ovation™ Whole Blood Solution.
In general any method that yields high quality, non-degraded RNA that is free of organic solvents and contaminants should work well.
Q1. 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 amplification.
Q2. 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 and a real time PCR system may be used for quality control.
Q3. What additional consumables are required or useful for the Ovation™ RNA Amplification System V2?
Only lab consumables are required, see page 5 for a listing. For the optional cDNA purification, the Zymo Research DNA Clean and Concentrator™-25 columns are needed (Zymo Research, Cat. #D4005).