Cycle threshold (Ct) values of the reference gene, B2M, were utilized to normalize the Ct-values for each sample describing the MPV17 expression using the equation ΔCt=meanCt MPV17−meanCt B2MPV17−meanCt
PCR amplification was performed for each DNA sample described above using primers designed to target the O-chain gene cluster of AltDE and AltDE1.
In addition to the multiple measurement from different sections of each individual sample described above, the ratio of positively stained surface area and total surface area were measured using Image J (NIH software) from reconstruction images made with Photoshop (Adobe Inc).
The data from a series of m microarray experiments can be represented as an m × n gene expression matrix (see Table 1), where each row represents a sample described by the expression of n genes from one experiment.
The specific gene expression was normalized to the level of 18S in each sample as described previously [ 26], taking into account the fidelity of each PCR reaction.
For cell phenotyping, 500 cells in total were evaluated in each sample, as described before [21].
For a loading control, the levels of expression of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein was detected in each sample as described previously [19], [39].
Expression of β-galactosidase was quantified by adding 400 µl Z-buffer contaning 1 mg/ml ONPG to 100 µl of each sample as described previously [36].
The QuantiTect Reverse Transcription Kit (QIAGEN) was used to synthesize cDNA from 2 µg of RNA of each sample as described by the manufacturer.
5 µg of RNA was isolated from each sample as described above and was used to create first-strand cDNA using random hexamers and reverse transcriptase (Superscript II; Invitrogen, Carslbad, CA), according to the manufacturer's instructions.
