Ovation^® PicoSL WTA System V2

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer, 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.

Can I perform fewer than 4 reactions at a time?

We recommend a minimum batch size of four reactions. Smaller batch sizes may result in difficulty with pipetting small volumes, as well as obtaining fewer than 12 reactions in total.

Is the Ovation PicoSL WTA System V2 3´ biased?

In this system, oligo dT primers are mixed with random primers for the first strand synthesis of cDNA products. This allows the product to be analyzed on 3‘ expression arrays and whole transcriptome arrays when used with an appropriate Encore labeling module, or other supported labeling protocol. Additionally, the random primers allow the detection of the entire transcripts when used as a pre-qPCR amplification system.

What methods do you recommend for RNA isolation?

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 extractions 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?

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, lower quality RNA samples and transcripts with a compromised poly(A) tail can also be amplified successfully using the Ovation PicoSL WTA System V2. The RNA should have high purity, however, and be free of contaminants.

Can contaminating genomic DNA interfere with the amplification performance?

Yes. This system is designed to amplify RNA, but contaminating genomic DNA may amplify during the process. For this reason we recommend DNase treatment during RNA purification.

Do you recommend DNase treatment of my total RNA sample?

Yes. For an explanation of DNase requirements see section III.A.5 of the User Guide. You may also find recommended procedures for DNase treatment in Appendix D.

Are there any tissues that will not work with the Ovation PicoSL WTA System V2?

We have not encountered any specific RNA sources that will not work with the Ovation PicoSL WTA System V2. The RNA should have high purity and be free of contaminants.

How many rounds of amplification are performed with the Ovation PicoSL WTA System V2?

This System has a single round of amplification. It cannot be used for multiple rounds.

What purification methods do you recommend?

• For the Second Strand cDNA purification step (pre-amplification) we require the use of the Agencourt Beads provided with the kit.
• Several purification options are available for the final SPIA cDNA cleanup step. These are described in Appendix A 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?

The SPIA cDNA can be stored at -20°C prior to performing the purification. We do not recommend stopping at any intermediate stage of the protocol.

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP 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 maximum 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 maximum 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.
• Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

How much cDNA can I expect from a single reaction?

You should expect 2–4 μg of cDNA from input of 500 pg to 50 ng total RNA.

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 PicoSL WTA System V2?

The SPIA cDNA size distribution is somewhat dependent on the input RNA integrity. A representative size distribution is given in Figure 4.

How do I measure my SPIA cDNA product yield?

You may use a Nanodrop, Qubit, or standard spectrophotometer. Refer to Section IV, Protocol I in the User Guide for specific guidance.

How many qPCR reactions will I get from one Ovation PicoSL WTA System V2 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 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 SPIA cDNA prior to qPCR analysis.

Do you recommend purification of the cDNA prior to qPCR analysis?

Yes. Although this is not absolutely necessary, it is important to be able to quantify the SPIA cDNA. This allows assessment of amplification success based on the amplification yields. It also allows mass normalization of the cDNA into qPCR.

Where in my target sequence can I design my qPCR primers?

The Ovation PicoSL WTA System V2 does not have a 3’ 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.