Transplex™ Whole Transcriptome Amplification FAQs

• How does WTA work?
• What type of RNA may be used?
• How much RNA is required to successfully perform WTA amplification?
• Can WTA be used to amplify archival fixed tissue or degraded samples?
• How can I optimize amplification yields?
• Once library amplification is complete how should the samples be purified?
• I need to quantify my WTA product, what is the preferred method?
• What are the downstream applications of WTA products?

1. How does WTA work?
   The WTA kit has two steps, library synthesis and library amplification. The library is synthesized when sample RNA is incubated with a reverse transcriptase and non-self-complementary primers comprised of a quasi-random 3’ end and a universal 5’ end. When annealed primers are extended by polymerase, displaced single strands are generated which become new templates for primer annealing and extension. This process creates an OmniPlex^® library comprised of random, overlapping 100-1000 base fragments flanked by a universal end sequence. Universal-primer PCR is then used to amplify the OmniPlex^® library and produce WTA products.
   
   
2. What type of RNA may be used?
   RNA can be isolated using standard methods or kits; RNA from numerous source materials may be used including blood, tissue biopsy, cultured cells and fixed or frozen tissues. Non-human sources of RNA may also be used such as animals, plants, or microorganisms.
   
   
3. How much RNA is required to successfully perform WTA amplification?
   For the most robust performance there should be at least 50 ng of RNA at a concentration of >5 ng/µl in TE, samples of RNA containing < 5 ng are useable. The RNA can be single-stranded or double-stranded and should have a molecular weight of at least 300 bases.
   
   
4. Can WTA be used to amplify archival fixed tissue or degraded samples?
   Yes, the WTA kit effectively amplifies degraded RNA, including formalin-fixed and paraffin-embedded samples. However, to acceptably amplify the final product, degraded samples require more starting material, but no more than 300 ng should be used.

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5. How can I optimize amplification yields?
   Optimal PCR cycle numbers might vary with template amount and quality. 17 cycles are recommended for 5 ng library aliquots of high quality RNA. If using a real-time system, the optimal cycle number is defined as the last cycle of a 2-3 cycle “plateau” phase in which the relative fluorescence unit stays constant.
   
   
6. Once library amplification is complete how should the samples be purified?
   Upon completion of library amplification the cDNA should be purified to remove residual primers and nucleotides that may interfere with downstream applications. We recommend the Sigma-Aldrich GenElute PCR DNA Purification Kit (NA1020) for the purification of single-stranded and/or double stranded amplification products from other reaction components such as excess primers, nucleotides or polymerases.
   
   
7. I need to quantify my WTA product, what is the preferred method?
   UV absorbance (A₂₆₀) should be used to quantify purified products using the conversion of 1 OD = 50 µg/ml. PicoGreen^® should not be used for quantification because it cannot efficiently detect single-stranded products and will underestimate the DNA yield. Each aliquot of library generated from high quality human total RNA will generate between 4 to 8 µg of WTA product. If electrophoresed, the product appears as a smear with a size distribution of 0.2 kb to 2 kb on a 0.8% agarose gel. Yield and size distribution of products may vary depending on the integrity and purity of the sample RNA.
   
   
8. What are the downstream applications of WTA products?
   WTA amplification forms a faithful cDNA library of your RNA template by amplifying the cDNA in a single PCR reaction. Reagents and protocols have been optimized to give linear representation of all expressed genes and exons. Depending on the amount of total RNA amplified WTA yields between 10-200 µg of amplified cDNA without compromise of quality. Upon creation of a cDNA library quantitative qPCR or microarray analysis may be performed.
   
   

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