Applause® WT-Amp Plus ST System
The Applause® WT-Amp Plus ST System provides a low-cost, single-tube, single purification, add-and-incubate workflow for whole transcriptome cDNA generation from 50 – 200 ng total RNA. The cDNA generated from the system is suitable for use with whole transcript array designs. The Applause® WT-Amp Plus ST System is powered by Ribo-SPIA technology, a rapid and sensitive amplification process developed by NuGEN.
Complete workflow in a single day
- Simple add and incubate in a single tube
- No additional ribosomal reduction step required
- cDNA ready to label in about 5 hours
- Amplified cDNA can be fragmented and labeled in less than 2 hours using NuGEN’s Encore® Biotin Module
Obtain consistent results
- Demonstrated robustness across a range of sample types
- Consistent data with a high degree of sensitivity, specificity and reproducibility
- Gene expression
- Sample archiving
|Compatible Platforms||Affymetrix GeneChip Arrays|
|Amplification Type||Whole transcriptome|
|Starting Material||Total RNA|
|Input Amounts||50 – 200 ng|
The Applause WT-Amp Plus ST System provides all necessary buffers, primers and enzymes for first strand cDNA synthesis, second strand cDNA synthesis and amplification, and all necessary buffers and enzymes for converting amplified cDNA into sense target cDNA (ST-cDNA). For your convenience, nuclease-free water has also been included.
What additional consumables does the user need?
For the ST-cDNA purification step, the QIAGEN MinElute Reaction Cleanup Kit, Catalog #28204, is required. The User Guide also lists recommendations for specific consumables, including nuclease-free pipette tips, nuclease-free microcentrifuge tubes, 0.2 mL PCR tubes and plates, RNaseZap and DNA-OFF.
What equipment is required or will be useful?
Required equipment includes a microcentrifuge, pipettes, vorteer, a thermal cycler, and a Nanodrop or UV/Vis spectrophotometer. An Agilent Bioanalyzer or a similar instrument may be used for quality control.
Does the Applause WT-Amp Plus ST System provide any labeling reagents?
No. The Applause WT-Amp Plus ST System is used to generate ST-cDNA from total RNA for use in gene epression eperiments. The resulting ST-cDNA may be processed further using the Encore Biotin Module for labeling and analysis on Affymetri GeneChip Gene 1.0 ST and Eon 1.0 ST Arrays.
What are the recommended storage conditions for the Applause WT-Amp Plus ST System components?
All components of the system may be stored at –20°C. Ensure the vials are well sealed and do not eceed 6 freeze/thaw cycles.
Has NuGEN performed reproducibility studies on the Applause WT-Amp Plus ST System?
Yes. Sample-to-sample, lot-to-lot and operator-to-operator reproducibility studies are routinely conducted according to NuGEN’s internal Quality Control metrics.
Can I use the Applause WT-Amp Plus ST System for archiving cDNA?
Yes. Amplified cDNA may be safely stored at –20°C for si months or longer.
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 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.
What is the minimum input required for amplification? Is there a maimum input?
The Applause WT-Amp Plus ST System can be used with high-quality, purified total RNA in the range from 50 to 200 ng. Input amounts outside this range may produce unsatisfactory and variable results.
Can I amplify degraded RNA with the Applause WT-Amp Plus ST System?
The Applause WT-Amp Plus ST System is not designed for use with degraded RNA. Using compromised samples will result in unsatisfactory and variable results.
Can DNA be used as input for the Applause WT-Amp Plus ST System?
No. The Applause WT-Amp Plus ST System is designed to amplify total RNA, not DNA.
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.
Does NuGEN recommend DNase treatment of purified total RNA samples?
Yes. For DNase treatment of RNA samples, refer to Appendi D of the User Guide.
Can I use the Applause WT-Amp Plus ST System on bacterial RNA samples?
The amplification process theoretically will work with many bacterial species; however, the kit has not been optimized for this purpose and NuGEN cannot guarantee success with such samples.
We recommend a minimum batch size of eight reactions. Smaller batches may result in poor performance due to the challenge of accurately pipetting small volumes.
How many rounds of amplification are performed in the Applause WT-Amp Plus ST System?
This System performs a single round of amplification. It is not designed to support multiple rounds of amplification.
Do I need to order specific primers for the amplification?
No. The DNA/RNA primers provided in the Applause WT-Amp Plus ST Systems are universal. No gene-specific primers are required.
Do I have to use the DNA/RNA primers supplied with the kit?
Yes. The Applause WT-Amp Plus ST System will not work properly with other primers.
Where can I safely stop in the protocol?
You may stop immediately following the SPIA Amplification protocol, or after Post-SPIA Modification II protocol prior to final cleanup at the points specifically noted in the protocol. Store reaction products at –20°C.
What are the recommended storage conditions for the amplified ST-cDNA?
The amplified ST-cDNA may be stored at –20°C.
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.
You should epect a minimum yield of 4 μg for the Applause WT-Amp Plus ST System when used as directed.
How do I quantitate the amplified cDNA product?
You may use a NanoDrop, Qubit or standard UV-Vis spectrophotometer. Be sure to use the single-stranded cDNA conversion factor of 1 A260 unit = 33 ng/μL in calculating the amplified cDNA concentration as this is the convention we used in establishing yield guidelines in the product User Guide.
Why do I need to use the single-stranded cDNA conversion factor when converting my A260 reading to cDNA concentration?
The amplified cDNA product of the Applause WT-Amp Plus ST Systems consists of both sense and antisense cDNA strands. While there may be some double-stranded character to this miture, we have developed and optimized the kit using the single-stranded cDNA conversion factor (1 A260 unit = 33 ng/μL of cDNA). Epected cDNA yield from amplification and input into labeling protocols cited in the product materials all have been generated using this convention.
No. The recommendations given in Appendi B of the User Guide describe the use of diluted, unpurified SPIA cDNA as the optimal template for qPCR reactions.
Where in my target sequence can I design qPCR primers?
The Applause WT-Amp Plus ST System amplifies the entire transcript 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.
How many qPCR reactions will I get from one Applause WT-Amp Plus ST amplification?
The number of qPCR reactions depends on the abundance level of the genes being interrogated and the size of the SPIA cDNA aliquot set aside for this purpose. For medium- to high-copy number genes, the cDNA may be substantially diluted. For very-low-copy number genes you may need to use more cDNA per reaction.
For research use only. Not for use in diagnostic procedures.