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

Optimized Total RNA (μg)

1 to 15

1 to 10

1 to 5

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

-

Methodology

-

Homopolymeric tail added

1

-

Purification

-

Spin cartridge 1

0.5

-

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

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

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