Quality Control

Quality control should take place at every step of the experimental process and only arrays that pass all quality criteria should go on to the analysis process. The following is a review of the quality metrics commonly used with GeneChip arrays (Figure 3.19).

Fig. 3.19 Quality metrics. Discrete measures give an indication of the progress of one step in the process. For example, Bio B reports the efficiency ofthe labelling and hybridization, but does not tell us anything about the RNA quality. Cumulative measures report the success of all previous steps in the process. For example, a percent-present measure in the normal range indicates the success of all previous steps in the

Fig. 3.19 Quality metrics. Discrete measures give an indication of the progress of one step in the process. For example, Bio B reports the efficiency ofthe labelling and hybridization, but does not tell us anything about the RNA quality. Cumulative measures report the success of all previous steps in the process. For example, a percent-present measure in the normal range indicates the success of all previous steps in the process: the RNA was of good quality, the hybridization and labelling worked well, and the software was applied properly. Divisions between quality measures are seldom this clear. For example, background primarily reports the hybridization reaction, but it is often influenced by sample quality (see Table 3.2 for descriptions of terms).

Tab. 3.4 Key quality-control measures.

Variable

Description

260/280 ratio (upon isolation)

The absorbance of the RNA should be measured at 260 and

280 nm to determine sample concentration and purity. The

260/280 ratio (upon processing)

A260/A280 ratio should be close to 2.0 for pure RNA (ratios

between 1.9 and 2.1 are acceptable).

Scale factor

When the two arrays are globally scaled to the same Target

Intensity, the scaling factor for a poorly hybridized sample

will be much higher than for a properly hybridized sample.

RawQ

A measure of the pixel-to-pixel variation of probe cells on a

GeneChip® array.

Background average

A measurement of signal intensity caused by auto-fluores-

cence of the array surface and non-specific binding.

Percent present

The number of probe sets called "Present" by the software

relative to the total number of probe sets on the array.

Actin 3' 5' ratio

Signal values of the 3' probe sets for actin and GAPDH are

GAPDH 3' 5' ratio

compared to the Signal values of the corresponding 5' probe

sets. The ratio of the 3' probe set to the 5' probe set is gene

rally no more than 3 for the 1-cycle assay.

BioB

BioB represents a gene in the biotin synthesis pathway of

E. coli. This transcript is added to the hybridization mixture

as a control.

IVTyield (micrograms cRNA)

The yield produced by the in vitro transcription (IVT) reac-

tion.

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