RNA yield will be quantified by a JENWAY Genova Spectrophotometer (A260).
In order to obtain sufficient quantity of labeled cRNA for target assessment
and hybridisation to GeneChip® expression probe arrays, we recommend
starting the analysis with:
RNA LabChip® Analysis
Size distrubution of purified cRNA will be assessed on
the Agilent Bioanalyzer 2100 using RNA 6000 Nano Assay, to ensure that
the cRNA amplification was successful before proceeding on to fragmentation
and hybridization.
Figure 3: A good cRNA reaction as represented
by a smear of products falling between 50 and 3000 bases long.
3rd QC - Validation of fragmented
cRNA
RNA LabChip® Analysis
Size distribution of fragmented labeled transcripts will be assessed
on the Agilent Bioanalyzer 2100 using RNA 6000 Nano Assay.
Figure 4: The standard fragmentation procedure
should produce a distribution of RNA fragment sizes from approximately
35-200 bases.
Following hybridization of fragmented cRNA to a GeneChip®
expression probe arrays, fluorescent signals are detected using a GeneArray™
scanner. Resulting array image is analysed by Affymetrix Microarray
Suite software (MAS 5.0) which generates an expression report file that
lists the quality control parameters. All of these parameters will be
scrutinised by an experienced Project Scientist to determine whether
array data has "passed" our quality standards:
Scaling Factor (SF) - For the
majority of the experiments where a relative small subset of transcript
is changing, the global method of scaling is recommended. The Target
Intensity (TGT) is an arbitrary figure set by the experimenter. A TGT
of 100 is a good value to use. The Scaling Factor (SF) is the multiplication
factor applied to the Average Intensity of an array to make it equal
to the Target Intensity, and thus make different experiments comparable.
A Scaling Factor of 1.0 would indicate that the Array Intensity is equal
to the Target Intensity. SF of 3-fold or more, indicates wide variation
in the unanalyzed data (i.e. the fluorescence intensities in the .dat
and .cel files), and therefore that the analyzed data (in the .chp file)
should be treated with caution.
Average Background
and Noise - Background value is a measure of the signal
intensity caused by auto fluorescence of the array surface as well as
nonspecific binding of target or stain molecules (SAPE). Noise (Q) is
a measure of the pixel-to-pixel variation of probe cells on a GeneChip®
array.
Percent Genes Present -
The number of probe sets called "Present" relative to the
total number of probe sets on the array is displayed as a percentage
(%P). Percent Present values depend on multiple factors including cell/tissue
type, biological or environmental stimuli, probe array type, and overall
quality of RNA.
The 5' and
3' Control Probe Sets - In addition to the conventional
probe sets, which are designed to be complementary to the region within
500bp of the 3' end of transcripts, the internal control genes (actin,
GAPDH, etc.) also have 5' and in some cases M (middle) probe sets included
on the arrays. These are intended to give an indication of the integrity
of the sample RNA, and efficiency of 1st strand cDNA synthesis. In general,
you would expect the Signal for the 3' probe sets to be somewhat higher
than for the M and 5' probe sets, because of the direction of synthesis.
A ratio of more than 3 for the 3'/5' signal values indicates a potential
sample problem.
Hybridisation Controls. bioB, bioC, bioD and
cre - These are added to the hybridization cocktail. bioB
at 1.5 pM is considered to be at a concentration near to the limit of
sensitivity of the assay, and may or may not be called Present. The
other controls should be called as Present, with increasing Signal values
- bioC (5 pM), bioD (25 pM) and cre (100 pM).
4th QC - Report File for Test
array
The GeneChip® Test3 Array provides convenient and
inexpensive means of determining the quality of labeled target and efficiency
of hybridization prior to analysis on GeneChip® expression arrays.
QC standards:
