[跳轉至內容](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/TW/zh) 產品 購物車0 TWZH 產品 產品應用服務資源支援 [登入 / 註冊](https://www.sigmaaldrich.com/oidc-sign-in) [訂單查詢](https://www.sigmaaldrich.com/TW/zh/order-lookup) [快速下訂](https://www.sigmaaldrich.com/TW/zh/quick-order) 購物車0 [Home](https://www.sigmaaldrich.com/TW/en)[Polymerase Chain Reaction Applications](https://www.sigmaaldrich.com/TW/en/applications/genomics/pcr)Integration of Sigma® TransPlex® WTA and Complete WTA2 Kits # Integration of Sigma® TransPlex® WTA and Complete WTA2 Kits with the Agilent Microarray Workflow Amplification products generated by the TransPlex® WTA and Complete WTA2 kits are suitable for microarray target for expression analyses, and can be readily integrated into existing Agilent workflows. The following modifications are required: - __The TransPlex WTA amplification product is double-stranded cDNA__. Labeling, hybridization, and wash procedures are preformed using the workflow outlined in the __Agilent Oligonucleotide Array-Based CGH for Genomic DNA__ __Analysis ULS Labeling for Blood, Cells, Tissues or FFPE (with a High Throughput option)__ (Version 3.4, July, 2012). - Fragmentation of the amplification product is not required. - __The Agilent Genomic DNA ULS™ (Universal Linkage System) Labeling Kit__ procedure (Agilent Cat. No. 5190-0419), allows for direct labelling of the microarray target, __preferable__ to enzymatic incorporation of modified nucleotides during or following amplification. ## Preparation of WTA Amplification Product for Labeling 1. Perform WTA amplification as described in the product bulletins for the TransPlex WTA or Complete WTA2 kits, found on the Sigma-Aldrich website. 2. Purify the amplification product using the [GenElute™ PCR Cleanup kit (Cat. No. NA1020)](https://www.sigmaaldrich.com/TW/en/product/SIGMA/NA1020) eluting with sterile RNase-/DNase-free water (Cat. No. [W4502](https://www.sigmaaldrich.com/TW/en/product/SIGMA/W4502) or [W1754](https://www.sigmaaldrich.com/TW/en/product/SIGMA/W1754)). Note 1.    Thirty microliters is the minimum elution volume Elute with 50 ul for maximum yield. Note 2. The capacity of the GenElute PCR Cleanup filter cartridge is 10 μg, sufficient for the typical yield of a __single__ Transplex WTA amplification reaction. Note 3. Divalent cation contamination (e.g. Mg 2+) will negatively affect ULS labelling efficiency. The GenElute PCR Cleanup kit adequately removes divalent cation contaminants and chaotropic salt contamination. 3. Determine concentration of purified amplification product using Nanodrop spectrophotometry. Use the following table (adapted from the Agilent procedure) to assure you have the appropriate DNA concentration for target labeling for the respective Agilent array formats Note 4. The DNA volume shown is required for subsequent steps in the Agilent workflow. Add nuclease-free water to bring to volume if necessary. Microarray format \*DNA input amount \[ng]† Volume of DNA \[μL] Minimum DNA concentration \[ng/μL] 1x microarray (non-FFPE samples)150016.591 1x microarray (FFPE samples)200016125 2x microarray10001759 4x microarray500862.5 8x microarray250832 \* Input DNA requirements and volumes are the same for both FFPE and non-FFPE samples for the 2x, 4x, and 8x arrays. † You can use more DNA, but you will also need to use more ULS dye. Always use a ratio of 1 μL ULS dye per microgram DNA (see procedure below). 4. If concentration of the amplification product is required, use vacuum-centrifugation to avoid loss of amplified product. 5. __Fragmentation__ of WTA amplification product is __unnecessary.__ ## Entry into Agilent Workflow 1. Enter Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis ULS Labeling for Blood, Cells, Tissues or FFPE at the step “ULS Labeling. 2. Proceed without deviation from the Agilent procedure, through the following steps: ULS Labeling Removal of non-reacted ULS-Cy 3. Follow “Preparation of Labeled ‘Genomic' DNA for Hybridization” with the following modifications: Modification 1.  Do not add Cot-1 DNA to the Hybridization Master Mix.  Substitute with nuclease-free water.  (Cot-1 DNA blocking is unnecessary for transcriptome analyses.)      Modification 2.  In accordance with Modification 1 above, omit the 37 °C, 30-minute incubation. (This step is included to allow the Cot-1 DNA to hybridize to highly-repeated *genomic* target sequences before application to the array, for the purpose of reducing non-specific target-probe interaction. Since Cot-1 DNA is not added, omit this step.) Modification 3.  Before loading hybridization samples on the arrays, incubate the samples at 95 °C for 3 minutes.  Place immediately on ice until applying to the array.   Modification 4.  Hybridize for forty hours of 65 °C in all cases." ## Materials 抱歉，發生意外錯誤。 Response not successful: Received status code 500 ### References 1\. Ken Heuermann. Sr. Scientist, Life Science Division. 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