Wt ovation ffpe rna amplification system v2

To evaluate the quality of the amplified DNA obtained with WT-Ovation FFPE RNA Amplification System v2 (NuGEN) the expression of the RPL13a housekeeping gene was assessed using a TaqMan assay, which allows the amplification of a short sequence of 105 pb (Assay Id: Hs01926559_g1; Life Technologies). For each sample, 3.3 ng of amplified DNA were used in a 15-μl reaction volume, and the analysis was performed in triplicate. Real-time PCR was performed with the 7900HT Real-Time PCR System (Life Technologies) and the experiments were run using absolute quantification set-up. Data were analyzed by the SDS 2.4 software using a threshold of 0.2.
A small aliquot of DNA single strand product obtained with the WT-Ovation FFPE RNA Amplification System v2 was analyzed on the Agilent 2100 Bioanalyzer using the Agilent RNA 6000 NanoLabChip kit for an evaluation of amplicon size distribution. Only amplified DNAs with a significant amount of amplicons longer than 200 nt were used for the hybridization on Affymetrix; amplified DNAs with shorter amplicons were considered inadequate for further microarray analysis.

FFPE RNA (100 ng) in 5 μL of RNA-free water was used for retrotranscription into double-stranded cDNA and linearly amplified using the WT-Ovation FFPE RNA Amplification System v2 (NuGEN Technologies; San Carlos, CA) according to the manufacturer’s recommendations. To purify the cDNA the QIAquick PCR purification Kit (Qiagen, Valencia, CA) was used, with some modifications introduced by the NuGEN protocol.
To 160 μL of amplified cDNA, 800 μL of PB buffer were added. Thereafter the columns were washed 2 times, each with 700 μL of ethanol 80%.

There are at least three commercially available kits for amplifying several hundred picograms of total RNA to a certain amount of biotinylated cRNA that would be sufficient for the Illumina BeadChip–based microarray experiments: (1) TargetAmp 2-Round Aminoallyl aRNA amplification kit 1.0 (Epicentre Biotechnologies, Madison, WI, USA) with biotin-X-X-NHS (Epicentre Biotechnologies), (2) MessageAmp II aRNA amplification kit with biotin-11-CTP and biotin-16-UTP (Ambion, Austin, TX, USA), and (3) WT-Ovation FFPE RNA amplification system V2 (NuGEN, San Carlos, CA, USA). Six small aliquots (100 pg) of total RNA samples extracted from human peripheral blood leukocytes (Clontech Laboratories, Palo Alto, CA, USA) were amplified and biotinylated using each of the three kits in duplicates and applied to the Illumina Human-6v2 Expression BeadChips (Illumina, San Diego, CA, USA), according to the manufacturer's instructions. As a control, two standard amounts of aliquots (500 ng) of the same total RNA samples were subjected to biotinylated cRNA synthesis in duplicates following the standard protocol using the Illumina TotalPrep RNA amplification kit (Ambion) and applied to the same BeadChip microarray. We assessed the amplification efficiency of each kit, congruence between the duplicated microarray data, and linearity of the small amount- and standard amount-based microarray data.