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Table 3 Comparison of labeling methodologies for kits compared in this study.

From: Comparison of target labeling methods for use with Affymetrix GeneChips

Stage Steps One-Cycle Time (hours) BioArray Time (hours) Superscript Time (hours)
  Optimized Total RNA (μg) 1 to 15   1 to 10   1 to 5  
First Strand Synthesis Total RNA used 2.5 μg 1.5 2.5 μg 1.25 2.5 μg 2.5
  Primer T7-oligo(dT)   T7-oligo(dT)   T7-oligo(dT)  
  Primer annealing/RNA denaturation 70°C × 10' and 4°C × 2'   70°C × 10'   70°C × 10'  
  Reverse transcriptase Superscript II   Proprietary   Superscript III  
  Final reaction volume 20 μl   20 μl   20 μl  
  Incubation conditions 42°C × 1 h   42°C × 1 h   46°C × 2 h  
  Inactivate RT -   -   70°C × 10'  
  RNA elimination -   Base hydrolysis/Neutralization 0.5 -  
  Purification -   Spin cartridge 1 0.5 -  
Tailing and Termination Methodology -   Homopolymeric tail added 1 -  
  Purification -   Spin cartridge 1 0.5 -  
Second Strand cDNA synthesis Methodology Nick translation reaction 2.5 Extension reaction 1.25 Nick translation reaction 2
  Volume used from previous reaction All   All   All  
  Final reaction volume 150 μl   30 μl   150 μl in 2 tubes  
  Incubation conditions 16°C × 2 h   42°C × 1 h   16°C × 2 h  
  T4 DNA polymerase (sticky ends) 16°C × 5'   -   -  
  Stop reaction EDTA addition   -   -  
  Purification Spin cartridge 0.5 Spin cartridge 1 0.5 Spin cartridge 0.25
In vitro transcription amplification Volume used from previous reaction All 16.25 All 16.25 All 16.5
  Biotinylated nucleotide Pseudouridine biotinylated nucleotide   Biotin-11-UTP/Biotin-11-CTP   Biotin-11-UTP  
  Final volume IVT 42 μl   40 μl   40 μl  
  Incubation conditions 37°C × 16 h   37°C × 16 h   37°C × 16 h  
  DNAse I digestion -   -   37°C × 30'  
  Purification Spin cartridge 0.5 Spin cartridge 2 0.5 Spin cartridge 0.25
  Volume after purification 21 μl   60 μl   100 μl  
  Methodology Affymetrix   Affymetrix   Affymetrix  
Fragmentation protocol Amplified RNA used 20 μg 1.25 20 μg 1.25 20 μg 1.25
  Final volume Fragmentation 40 μl   40 μl   40 μl  
  Total Hours   22.5   23.5   22.75
  Number of steps 7   11   6  
  1. All details are taken from manufacturers manuals supplied with each kit.