Ovation® qPCR System
The Ovation® qPCR System provides a fast and simple method for preparing amplified cDNA from total RNA for gene expression analysis using real-time quantitative PCR (qPCR). Amplification is initiated at the 3´ end as well as randomly throughout the whole transcriptome. The Ovation® qPCR System is powered by Ribo-SPIA® technology, a rapid, simple and sensitive RNA amplification process developed by NuGEN.
- Simple workflow completed in about 4 hours using 5 -50 ng of input total RNA
- Amplified cDNA product is ready for qPCR and does not require purification
- Enables whole transcriptome gene expression analysis of compromised samples and detection of low-, medium- and high-abundance gene transcripts
The Ovation® qPCR System provides optimized reagent mixes and a protocol to process 24 samples.
Detect more genes with Ovation® qPCR System.
- Effective utilization of small and valuable samples
- Quantitative real-time PCR
- Gene expression
|Compatible Platform||All major qPCR machines|
|Amplification Type||Whole transcriptome|
|Starting Material||Total RNA|
|Input Amounts||5 – 50 ng|
The Ovation qPCR System provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and amplification, yielding cDNA. The kit also provides nuclease-free water.
What equipment is required or will be useful?
Required equipment includes a microcentrifuge, pipettes, vorteer and a thermal cycler. A UV/Vis spectrophotometer and an Agilent Bioanalyzer will be useful.
What additional consumables does the user need?
None. For the optional purification of amplified cDNA, see user guide for validated purification products and procedures.
Is the Ovation qPCR System 3´ biased?
No, this product is different from the Ovation RNA Amplification System in that the first strand cDNA is primed with random heamers as well as a 3´ primer.
What is the dynamic range of input mRNA that is amplified with the Ovation qPCR System?
Our studies demonstrate amplification over 6 orders of magnitude starting with transcripts present as low as 20 copies in a sample.
Has NuGEN performed reproducibility studies on the Ovation qPCR System?
Yes. Sample-to-sample, lot-to-lot and operator-to-operator reproducibility studies are routinely conducted.
Can I use the Ovation qPCR System for archiving cDNA?
Amplified cDNA may be stored at -20°C for at least several months.
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 etractions 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 do I need for amplification?
We recommend staying within the range of 5 to 50 ng total RNA starting material. Amounts greater than 50 ng may produce variable results.
Can the Ovation qPCR System amplify DNA?
The Ovation qPCR System is designed to amplify mRNA, not DNA.
Can I use the Ovation qPCR System on bacterial RNA samples?
The Ovation qPCR System theoretically will work with some bacterial RNAs. However, the kit has not been optimized for this purpose.
Are there any tissues that will not work with the Ovation qPCR System?
We have not encountered any good quality, clean RNA samples containing poly(A)+ RNA that will not work with the Ovation qPCR System.
We recommend 3 batches of 8 reactions. Smaller batch sizes may result in fewer than 24 reactions in total.
Does the Ovation qPCR System generate product in the absence of RNA input?
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 qPCR System?
The Ovation qPCR System performs a single round of amplification.
Do I need to order specific primers for the amplification?
No. The DNA/RNA primers provided in the Ovation qPCR System are universal.
Do I have to use the DNA/RNA primers supplied with the kit?
Yes. The Ovation qPCR System will not work properly with other primers.
If I choose to purify my product, what method do you recommend?
Several purification options are available for the final SPIA cDNA cleanup step. These are described in Appendi B of the User Guide. Selection of the optimum purification option can depend on many factors. Please contact the NuGEN Technical Support team for assistance in selecting the appropriate option for your application. Refer to section II.B for ordering information.
Where can I safely stop in the protocol?
We do not recommend stopping at any stage of the protocol.
Based on qPCR on a variety of genes, an average amplification efficiency of 1500-fold is observed.
How much cDNA can I epect from a single reaction?
You should epect 1.5 to 4 μg of cDNA from 5 to 50 ng total RNA starting material.
Is the cDNA yield dependent upon the quantity of total RNA input?
Yes, higher RNA inputs will produce higher yields. However, at inputs of above 50 ng, the yields may become variable without increasing.
What size cDNA is generated by the Ovation qPCR System?
On a Bioanalyzer, using the RNA 6000 size markers, the average size of the amplified cDNA is 375 bases. More than 50% of the product is greater than 320 bases 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.
The Ovation qPCR System does not have a 3 prime bias 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.
How many qPCR reactions will I get from one Ovation qPCR System 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. You will need to determine how much cDNA to use per reaction depending on the abundance of the gene being interrogated.
For research use only. Not for use in diagnostic procedures.