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 a library comprised of random, overlapping 100-1000 base fragments flanked by a universal end sequence. Universal-primer PCR is then used to amplify the library and produce WTA products.
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.
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.
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.
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.
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.
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.
WTA amplification creates a cDNA library of the RNA template. Applications such as qPCR, traditional cloning (TOPO TA Cloning®), micro array may be performed.
The WGA2 provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and cDNA library amplification.
A microcentrifuge, pipettes, vortexer, a thermal cycler, a spectrophotometer are required, an Agilent Bioanalyzer® may also be useful.
No, in the system, quasi-random WTA primers with universal ends are used for synthesis of cDNA products. This enables the amplification of highly degraded RNA in which much of the amplifiable sequence has become separated from the poly-A sequence. qPCR and microarray testing of both degraded and intact RNA vs 3' biased methods have been successful.
The system WTA2 includes random priming 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. We strongly recommend designing your assays for multiple locations across the transcript since the starting FFPE RNA is likely to be highly degraded.
Yes, the protocol is optimized to create microgram quantities of cDNA for microarray analysis. Scaling back the reaction volumes will not impact the quality of the results, only the quantity of final product.
The cDNA product is similar to any PCR product. A TOPO TA cloning method is suggested. Ensure a dilution is set-up to achieve the optimal cloning results.