Integration of Sigma® TransPlex® WTA and the Complete WTA2 Kits with the Affymetrix Microarray Workflow

Amplification products generated by the TransPlex® WTA and Complete WTA2 kits are suitable for microarray target for expression analyses, and can be incorporated into existing Affymetrix workflows through a procedure known as Pico Profiling1,2. Pico Profiling is comprised of a modified Affymetrix GeneChip® Mapping 10K Assay Protocol3 for fragmentation and labeling of the amplification product, followed by a modified hybridization procedure taken from the Affymetrix GeneChip® Expression Analysis Technical Manual4.

The WTA amplification product is double-stranded cDNA. Fragmentation and labeling is accomplished using the following Affymetrix products.

  • GeneChip Fragmentation Reagent (Affymetrix product # 900131, made to order)
  • 10X Fragmentation Buffer (Affymetrix product # 900422).
  • Terminal Deoxynucleotidyl Transferase (rTdT), Recombinant, 500 units, with buffer (Affymetrix product # 72033 500 UN)
  • Biotin-11-dXTP Analog (DNA Labeling Reagent, DLR), 250 nmol, 10 mM (Affymetrix product # 79015 250 NM)

Preparation of WTA Amplification Product for Fragmentation and 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 (Sigma Cat. No. NA1020, GenElute PCR Cleanup Kit), eluting with sterile RNase-/DNase-free water (Sigma Cat. No. W4502 or W1754).

    Note 1.  Elute with 30 - 50 μl nuclease-free water. Thirty microliters is the absolute minimum elution volume,
                   for highest potential concentration. Elute in 50 μL 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.

  3. If necessary, adjust the concentration of the amplification product to > 0.36 μg/μL, using vacuum-centrifugation to avoid loss of amplified product. Determine DNA concentration using Nanodrop spectrophotometry.

Entry into Pico Profiling Workflow

WTA RNA Amplification and Pico Profiling

The Transplex WTA amd  Complete WTA2 RNA amplification kits are designed to representatively amplify RNA of marginal quality, such as degraded RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples or small quantities of RNA damaged by laser capture. In addition, the kit is equally suitable for high quality RNA. Pico Profiling3,4 is a complete process for isolation and purification of total RNA from single or very small numbers of cells, followed by Complete WTA2 amplification and analysis on Affymetrix expression microarrays. Moreover, Pico Profiling steps subsequent to RNA isolation and purification can be used for any quantity of input RNA.

When working with small RNA samples, follow the Pico Profiling protocol in its entirety; otherwise enter the protocol following amplification at the step "cDNA Fragmentation”. Follow procedure presented on the Institute for Research in Biomedicine (IRB, Barcelona) Functional Genomics Core Facility website as outlined.2

Enter the Pico Profiling protocol, version 02/11, at “cDNA Fragmentation”

Follow procedure presented on the Institute for Research in Biomedicine (IRB, Barcelona) Functional Genomics Core Facility website as described.3

Note the following points:

  1. GeneChip Fragmentation Reagent (Affymetrix product # 900131, substitute for # 901010) and 10X Fragmentation Buffer (Affymetrix product # 900422) are components in the Genechip? Mapping 10K Xba Assay kit (#900441, MTO) available upon inquiry from Affymetrix.

  2. Reaction volumes should be scaled to accommodate requirements for the specific microchip used. However, use 8 to 10 mg cDNA target regardless of hybridization volume.2

  3. The fragmentation step has been rigorously optimized by Affymetrix. However it is critical that the Fragmentation Reagent is diluted correctly. Fragmentation should produce an Agilent Bioanalyzer peak of 40 to 70 base-pair fragments, with an overall range of 20 to 200 base pairs.2

  4. Terminal transferase labeling should be qualitatively assayed before proceeding to hybridization, utilizing the “Gel Shift” procedure described in Chapter 4 of the Affymetrix GeneChip® Expression Analysis Technical Manual.

  5. For hybridization, follow the Pico Profiling procedure, a modification of the Affymetrix GeneChip® Expression Analysis Technical Manual.

Materials

     

References

  1. Eva Gonzalez-Roca, Xabier Garcia-Albéniz, Silvia Rodriguez-Mulero, Roger R. Gomis, Karl Kornacker, Herbert Auer. 2010. Accurate Expression Profiling of Very Small Cell Populations. PLOS One 5 (12): e14418.
  2. Pico Profiling, v. Feb, 2011. Institute for Research in Biomedicine (Barcelona) Functional Genomics Core Facility website: http://www.dnaarrays.org.  Downloads: http://www.dnaarrays.org/D_FileS1Feb8.pdf.
  3. Affymetrix GeneChip® Mapping 10K 2.0 Assay Manual, P/N 70722 Rev. 3, ©2004 Affymetrix, Inc.
  4. Affymetrix GeneChip® Expression Analysis Technical Manual, P/N 702232 Rev. 3, ©2005-2009 Affymetrix Inc.

 

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