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Improved data normalization methods for reverse phase protein microarray analysis of complex biological samples
 
Antonella Chiechi1,2, Claudius Mueller2, Kevin M. Boehm2, Alessandra Romano2, Maria Serena Benassi1, Piero Picci1, Lance A. Liotta2, and Virginia Espina2
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Supplementary Material
Protocol (.pdf)

Selection of analytes for RPMA normalization

The suitability (stability) of seven normalization analytes (ssDNA, total protein, GAPDH, MRPL11, RPL13a, α/β-tubulin, and β-actin) and 13 protein analytes (Akt Ser473, Akt Thr308, BAD Ser112, p38 Thr180, Cleaved Caspase 3 Asp198, EGFR Tyr1148, GSK3 a/b Ser219, IKappaB Ser32, IRS1 Ser612, MARCKS Ser152/156, ERK Thr202/Tyr204, CREB Ser133, and STAT1 Tyr701) was evaluated by geNorm (7) and NormFinder (8).

Statistics

The Mann-Whitney t-test on medians (GraphPad Prism v 5.03, GraphPad Software, Inc.) was applied to groups of samples from the multiple myeloma core biopsy data set normalized by ssDNA, total protein, and β-actin for comparison of three normalization methods.

Results and discussion

Normalization of spot intensity values is a major issue in microarray data analysis. A priori selection of the normalization parameters may not take into account potential sources of contamination and/or variability. In the present study, we introduce the novel use of ssDNA alone or in combination with cellular proteins as normalization molecules which can aid in selecting the optimal normalization parameter for a given RPMA study set.

In order to standardize and streamline data analysis, we have automated part of the data reduction/normalization processes by writing a VBA Excel macro, RAS. RAS was designed to standardize the data reduction steps after image acquisition and raw pixel intensity generation, while permitting flexibility in RPMA array design and normalization parameters. RAS operational features are: (i) removal of flagged spots from the downstream analysis; (ii) correction of pixel intensities below zero; (iii) quality control filters based on replicate spot CV and spot intensity versus background; (iv) subtraction of non-specific signal; and (v) normalization to user-specified endpoints or the geometric mean of several endpoints.

To measure nucleic acids (ssDNA) or proteins on a nitrocellulose membrane, we developed a microarray treatment strategy to ensure ssDNA binding without disrupting protein binding. Nitrocellulose does not bind double-stranded DNA (14, 15) but ssDNA provides an ionic interaction between negatively charged phosphate groups of the nucleic acid and the positively charged nitrocellulose. DNA in our protein whole cell lysates assumes the single-stranded conformation because denaturing reagents (TCEP and SDS) used in the protein extraction buffer and heating cause DNA strand scission (16, 17). The first RPMA processing modification we made was to eliminate alkaline pre-treatment (ReBlot) of the RPMA prior to immunostaining for any microarray that was destined to be stained with anti-ssDNA. Alkaline pre-treatment is often performed to ensure that the immobilized proteins are fully denatured, which provides optimal linear protein conformation for antibody binding. However, alkaline solutions can remove DNA from the nitrocellulose due to strong charge interactions (14, 15) between the alkali and the DNA.

The second RPMA processing modification we made was to ensure that the ssDNA was bound tightly to the nitrocellulose. After printing, baking the RPMA nitrocellulose slide at 80°C for 2 h ensures that the ssDNA fragments become firmly attached to the nitrocellulose matrix (15).

The effect of baking on protein antigenicity could hypothetically affect immunostaining. We performed a series of replicate experiments to compare antibody binding/reactivity on baked and unbaked reverse phase protein microarrays. A total of 74 identical arrays (37 baked and 37 non baked), containing ssDNA as a positive control, RNA as a negative control, and several different cell and tissue lysates, were stained with Sypro Ruby for total protein (Supplementary Figure S2A), probed with antibodies directed against a phosphorylated protein (phospho-Akt Ser473; Supplementary Figure S2B), or non-phosphorylated protein (β-actin; Supplementary Figure S2C). Baking reverse phase protein microarrays retains protein antigenicity/reactivity as shown by equivalent staining intensity between baked and unbaked array slides.

The third RPMA processing modification was to optimize a detection method for ssDNA. The most common fluorescent dyes for DNA detection, such as DAPI, SYBR GREEN II, and acridine orange (18, 19), cannot discern DNA from RNA and the autofluorescence of nitrocellulose overlaps with the emission wavelength of each of these dyes (20).

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