Ovation® One-Direct System
The Ovation® One-Direct System provides a fast and simple method for preparing amplified cDNA directly from cell lysate from few or single cells as well as 10-500 pg of purified total RNA samples. Powered by NuGEN’s Ribo-SPIA technology, amplification is initiated at the 3’ end as well as randomly throughout the whole transcriptome. The amplified cDNA is suitable for multiple downstream applications including microarrays, qPCR or sample archiving.
Streamline your workflow
- Simple workflow that begins directly with cell lysates with no RNA purification required.
- Single day protocol that can be completed in about 8 hours
- Amplified cDNA can be fragmented and labeled in less than 2 hours using either NuGEN’s Encore® Biotin or BiotinIL Module
- Gene expression
- Single Cell
|Compatible Platform||Affymetrix GeneChip Arrays, Illumina BeadChips, Agilent Dual Mode Arrays|
|Amplification Type||Whole transcriptome|
|Starting Material||Cell lysate or total RNA|
|Input Amounts||10 - 500 pg|
Required equipment includes a microcentrifuge, pipettes, vorteer, a thermal cycler, a spectrophotometer and a magnetic plate for 0.2 mL tubes, strips, or plates. An Agilent Bioanalyzer or Tapestation may also be useful for optional analytical tests.
Does the Ovation One-Direct System provide any fragmentation and labeling reagents?
No. The Ovation One-Direct System is used to generate cDNA from cell lysate or ultra-small amounts of total RNA for use in gene epression eperiments. The resulting cDNA may be processed further using the Encore Biotin Module for labeling and analysis on Affymetri GeneChip arrays, or processed for use on Agilent, or Illumina microarrays using other supported labeling protocols.
Can the Ovation One-Direct System be used for Net Generation Sequencing (NGS) applications?
This amplification system is generally not advisable for use in NGS workflows. Please contact NuGEN Technical Support to discuss your specific needs for NGS sample preparation.
We do not recommend the use of TRIzol® or similar methods as any carry over of organics may inhibit downstream enzyme activity. If using TRIzol etracted 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.
Can contaminating genomic DNA interfere with Ovation One-Direct System performance?
Yes. When using purified total RNA samples, ensure that the samples have a minimum amount of contaminating genomic DNA. In high quantities, genomic DNA will interfere with mRNA amplification.
Are there any tissues that will not work with the Ovation One-Direct System?
We have not encountered any specific RNA sources that will not work with the Ovation System. Some cell types may not be suitable for lysis using the Direct Lysis Buffer.
Can DNA be used as input for the Ovation One-Direct System?
No. The Ovation One-Direct System is designed to amplify total RNA, not DNA.
Cell lysates can be stored at –80°C for up to 2 weeks. We do not recommend refreezing a lysate after its first thaw. Lysates from low numbers of cells may be less stable. For best performance we recommend amplifying such samples as soon as possible after lysis.
Do I need to order specific primers for the amplification?
No. The DNA/RNA primers provided in the Ovation One-Direct System are universal. No gene-specific primers are required.
What purification methods do you recommend?
For the double stranded cDNA purification step (pre-amplification) we require the use of the Agencourt Beads provided with the kit. For the amplified cDNA purification step we recommend using the QIAGEN MinElute Spin Column. Alternative methods are described in Appendi C of the User Guide.
RNAClean P beads are certified to be RNase and DNase free. We have tested both RNAClean P and AMPure P 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 maimum 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 maimum 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.
- Mi the bead suspension and sample thoroughly to ensure maimum binding of the samples to the beads.
We have observed yields of 7 μg or more of cDNA from amplification of 50 pg purified total RNA. Yields may be as high as 15 μg or more when RNA inputs of 100 pg to 500 pg are used.
How do I quantitate the amplified cDNA product?
You may use a Nanodrop, Qubit, or standard UV/Vis spectrophotometer (see section IV. J. in the User Guide for details). Be sure to use the single-stranded cDNA conversion factor of 1 A260 unit = 33 ng/μL in calculating the amplified cDNA concentration.
Can I use an Agilent Bioanalyzer, Bio-Rad Eperion, or other comparable instrument, to evaluate the amplification products?
Yes. Refer to Appendi D in the User Guide for guidelines.
The Ovation One-Direct System amplifies the whole transcriptome so primers can be designed at any location within the mRNA. In order to avoid interference from possible genomic DNA contamination, we recommend treating RNA with DNase and designing amplicons to span an intron.
For research use only. Not for use in diagnostic procedures.