Frequently Asked Questions
WT-Ovation™ FFPE RNA Amplification System V2
(Cat # 3400-12, 3400-60)
Q1. What materials are provided with the WT-Ovation™ FFPE RNA Amplification System?
The WT-Ovation™ FFPE 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™ FFPE RNA Amplification System provide any fragmentation and labeling reagents?
No, however the cDNA output of this kit may be processed further using validated NuGEN fragmentation and labeling products and protocols.
Q3. What equipment is required or will be useful?
A microcentrifuge, pipettes, vortexer, a thermal cycler, a spectrophotometer, and a magnetic plate are required, an Agilent Bioanalyzer may also be useful.
Q4. What additional consumables does the user need?
Purification columns and or beads.
Q5. Do I need to use high quality total RNA?
The WT-Ovation FFPE System V2 employs the whole transcriptome amplification approach and was designed and optimized for use with highly degraded, lower quality RNA samples and transcripts with a compromised poly-A.
Q6. Can I use RNA from other sources than FFPE?
Yes. Degraded as well as intact RNA from other sources may be successfully amplified using the WT-Ovation FFPE System V2. With good quality RNA we recommend you use 2-20 ng starting material.
Q7. Is the WT-Ovation™ FFPE System V2, 3 prime biased?
In this system, oligo dT primers are mixed with random primers for the first strand synthesis of cDNA products. This enables the amplification of highly degraded RNA in which much of the amplifiable sequence has become separated from the poly-A sequence. We have tested the system with both degraded and intact RNA on 3’ biased microarrays as well as arrays interrogating sequence in all regions of the transcript (i.e. “exon” arrays) with successful results.
Q8. Where in my target sequence can I design my qPCR primers?
WT-Ovation System V2 includes random priming and 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 your amplicons to span an intron. We strongly recommend designing your assays for multiple locations across the transcript since the starting FFPE RNA is likely to be highly degraded.
Q9. How much FFPE total RNA do I need for amplification?
We recommend total FFPE RNA inputs in the range of 50 ng to 100 ng. Input amounts outside this range may produce unsatisfactory variable results, especially for more degraded RNA.
Q10. How much cDNA can I expect from a single reaction?
You should expect 4 to 7 µg of cDNA from 50 -100 ng total FFPE RNA starting material, if it is of sufficient quality. Although yield is a critical sample quality indicator, success of a given FFPE sample set in array analysis maybe predicted using the RNA Sample Quality Assessment Tool described in WT-Ovation FFPE System Technical Report #1.
Q11. Is the cDNA yield dependent upon the quantity of total RNA input?
Yes, the higher the RNA input into the amplification reaction, the higher the yield will be. However, at FFPE RNA inputs of above 100 ng, the yields may become variable.
Q12. What is the amplification efficiency of the WT-Ovation™ FFPE System V2?
Based on qPCR on a variety of genes, an average amplification efficiency of 10,000 to15,000 fold is observed.
Q13. What size cDNA is generated by the WT-Ovation™ FFPE System V2?
The amplified cDNA size distribution is entirely dependent on the input RNA integrity. In a whole transcriptome amplifications strategy however, the size of the resulting cDNA is not of significant consequence for use on arrays.
Q14. Can DNA be used as input for the WT-Ovation™ FFPE System V2?
No. The Ovation™ FFPE System V2 is designed to amplify mRNA, not DNA.
Q15. Can contaminating genomic DNA interfere with the WT-Ovation™ FFPE System V2?
This system is designed to amplify RNA, but large amounts of contaminating genomic DNA may amplify during the process, so we recommend DNase treatment during RNA purification.
Q16. Can I use the WT-Ovation™ FFPE System V2 on bacterial RNA samples?
The WT-Ovation amplification process theoretically will work with some bacterial RNAs. However, currently, the kit has not been optimized or validated for this purpose.
Q17. Has NuGEN performed reproducibility studies on the WT-Ovation™ FFPE System V2?
Yes. Sample to sample, and lot to lot reproducibility studies are routinely conducted.
Q18. Does the WT-Ovation FFPE System V2 generate product in the absence of RNA input?
In the complete absence of input RNA non-specific product is generated with less than 3 µg yields. However, note that in the presence of even very small amount of RNA, while the yields may be low the cDNA is likely specific, and representing actual amplification products.
Q19. Can I use the WT-Ovation FFPE System V2 for archiving cDNA?
Amplified cDNA may be stored at -20°C for at least 6 months.
Q20. Do I need to order specific primers for the amplification?
No. The DNA/RNA primers provided in the WT-Ovation™ FFPE System V2 are universal.
Q21. Do I have to use your DNA/RNA primers?
Yes, the system will not perform with other primers.
Q22. 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.
Q23. What purification methods do you recommend?
For the double stranded cDNA purification step (pre-amplification) we require the use of the Agencourt RNAClean magnetic beads provided with the kit. For the amplified cDNA purification step we recommend the Zymo Research DNA Clean & Concentrator-25.
Q24. Where can I safely stop in the protocol?
We do not recommend stopping at any intermediate stage of the protocol.
Q25. Do you recommend DNase treatment of my total RNA sample?
Yes, see the Appendix section of product user guide.
Q26. How many qPCR reactions will I get from one WT-Ovation™ FFPE 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 thousands of qPCR reactions. For very low copy genes more cDNA must be used per qPCR reaction. We recommend purification of the amplified cDNA prior to qPCR analysis.