Frequently Asked Questions

WT-Ovation™ Pico RNA Amplification System
(Cat # 3300-12)

Q1. What materials are provided with the WT-Ovation™ Pico RNA Amplification System?
The WT-Ovation™ Pico System provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and amplification, yielding single stranded amplified cDNA. The kit also provides nuclease-free water and Agencourt® RNAClean® magnetic beads for double stranded cDNA purification.

Q2. Does the WT-Ovation™ Pico RNA Amplification System provide any fragmentation and labeling reagents?
No. The WT-Ovation™ Pico System is used to generate single stranded cDNA from small amounts of total RNA for qPCR analysis or archiving. However the cDNA output of this kit may be processed further using other validated NuGEN kits such as the FL-Ovation™ cDNA Biotin Module V2 kit in order fragment and label the cDNA and further analyze it on GeneChip® arrays.

Q3. What equipment is required or will be useful?
Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, and an O.D. spectrophotometer. An Agilent Bioanalyzer may be useful for optional analytical tests.

Q4. What additional consumables does the user need?
For the SPIA™cDNA purification step, purification columns are required, see user guide for validated purification products and procedures.

Q5. Do I need to use high quality total RNA?
The RNA should have high purity however and be free of contaminants. RNA samples of high molecular weight with little or no evidence of degradation, as expected, will amplify very well with this product. However, due to the whole transcriptome amplification approach, even lower quality RNA samples and transcripts with a compromised polyA can also amplify successfully.

Q6. Can I do reactions in smaller batches than 4?
We recommend 3 batches of 4 reactions. Smaller batch sizes may result in difficulty with pipetting small volumes as well as obtaining fewer than 12 reactions in total.

Q7. Is the WT-Ovation™ Pico System 3 prime biased?
No, this product is different from the Ovation™ RNA Amplification System in that the first strand cDNA is primed with random hexamers as well as a 3 prime primer.

Q8. Where in my target sequence can I design my qPCR primers?
The WT-Ovation™ system amplifies across the whole transcriptome, therefore primers can be designed at any location within the mRNA. In order to avoid qPCR interference from possible genomic DNA contamination, we recommend treating your RNA with DNase and designing amplicons to span an intron.

Q9. How much total RNA do I need for amplification?
We recommend staying within the range of 500 pg to 50 ng total RNA as starting material. Input amounts greater than 50 ng may produce variable results.

Q10. How much cDNA can I expect from a single reaction?
You should expect 6 to 10 μg of cDNA from input of 500 pg to 50 ng total RNA starting material.

Q11. Is the cDNA yield dependent upon the quantity of input total RNA?
Yes, the more RNA is put into the amplification reaction, the more yield is recovered. However at inputs of above 50 ng, the yields may become variable without increasing.

Q12. What is the amplification efficiency of the WT-Ovation™ Pico System?
Based on qPCR on a variety of genes, an average amplification efficiency of 10 to15 thousand fold is observed.

Q13. What size cDNA is generated by the WT-Ovation™ Pico System?
On a Bioanalyzer, using the RNA 6000 size markers, the median length of the amplified cDNA is approximately 340 bases. More than 50% of the product is greater than 320 bases in length.

Q14. Can the WT-Ovation™ Pico System amplify DNA?
No. The Ovation™ Pico System is designed to amplify mRNA, not DNA.

Q15. Can I use the WT-Ovation™ Pico System on bacterial RNA samples?
The WT-Ovation™ amplification process theoretically will work with some bacterial RNAs. However, currently, the kit has not been optimized for this purpose.

Q16. Are there any tissues that will not work with the WT-Ovation™ Pico System?
We have not encountered any specific RNA sources that will not work with the Ovation™ System. The RNA should have high purity and be free of contaminants.

Q17. Has NuGEN performed reproducibility studies on the WT-Ovation™ Pico System?
Yes. Sample to sample, lot to lot, and operator to operator reproducibility studies are routinely conducted.

Q18. Does the Ovation™ System generate product in the absence of RNA input?
In the complete absence of input RNA non-specific product is generated with 1-2 μg yields. However, note that in the presence of even very small amount of RNA, while the yields may be low, it likely is specific and actual amplification product.

Q19. How many rounds of amplification are performed with the WT-Ovation™ Pico System?
This System performs a single round of amplification and can not be used for multiple rounds of amplification.

Q20. Can I use the WT-Ovation™ Pico System for archiving cDNA?
Amplified cDNA maybe safely stored at -20 °C for 6 months. Longer term storage tests are in progress.

Q21. Do I need to order specific primers for the amplification?
No. The DNA/RNA primers provided in the WT-Ovation™ Pico System are universal.

Q22. Do I have to use your DNA/RNA primers?
The WT-Ovation™ Pico System will not work properly with other primers.

Q23. What are the incubation temperatures for each step?
First strand primer annealing = 65 °C
First strand synthesis = 4 °C, 25 °C, 42 °C, and 70 °C
Second strand synthesis = 4 °C, 25 °C, 50 °C, and 70 °C
SPIA™ amplification = 4 °C, 47 °C, and 95 °C

Q24. Do you recommend purification of the cDNA prior to qPCR analysis?
Yes. Although this is not typically necessary, it is important to be able to quantitate the amplified cDNA. This allows assessment of the amplification success based on the yields obtained, it also allows mass normalization of the cDNA into qPCR.

Q25. What purification methods do you recommend?

  • For the double stranded cDNA purification step (pre-amplification) we require the use of the Agencourt® RNAClean™ beads provided with the kit.
  • For the amplified cDNA purification step we highly recommend using the Zymo Research DNA Clean & Concentrator™-25. Alternative methods have also been validated and described in the user guide.
  • Refer to the user guide’s section Additional, Reagents, Supplies, and Equipment section, and the user guide Appendix for procedures and order information.

Q26. How do I measure my amplified cDNA product?
You may use a standard spectrophotometer or a Nanodrop.

Q27. Where can I safely stop in the protocol?
We do not recommend stopping at any stage of the protocol.

Q28. Do you recommend DNase treatment of my total RNA sample?
Yes. For an explanation of DNase requirements refer to the user guide Appendix. Procedural details and recommendations are listed in the user guide Appendix.

Q29. Where in my target sequence can I design my qPCR primers?
Since the WT-Ovation™ Pico RNA Amplification System is 3’ initiated as well as WT, qPCR assay primers can be designed at any location within the mRNA. In order to avoid qPCR interference from possible genomic DNA contamination, we recommend treating your RNA with DNase and designing your amplicons to span an intron.

Q30. How many qPCR reactions will I get from one WT-Ovation™ Pico amplification?
The number of qPCR reactions depends on the abundance level of the genes being interrogated. For medium to high copy genes, the cDNA may be diluted as much as 400-fold, enough for hundreds of qPCR reactions. For very low copy genes you will need to use more cDNA per reaction. The user will need to determine how much cDNA to use per reaction depending on the abundance of the gene being interrogated. Note that we recommend purification of the amplified cDNA prior to qPCR analysis. Note that we recommend purification of the amplified cDNA prior to qPCR analysis.