Core Director: Shixia Huang, Ph.D.

Technology & Infrastructure

Description of Technology

Reverse phase protein array (RPPA) is a high-throughput technology that performs protein assays on thousands of samples simultaneously, including tissue and cell lysates, serum, plasma or other body fluids. This protein array platform measures levels of protein expression, as well as protein modifications such as phosphorylation. Protein lysates are arrayed as microspots on nitrocellulized coated glass slides and probed with highly specific antibodies that have been validated for RPPA.  Each microspot contains the whole proteome repertoire of the tissue or cell. This enables the determination of a set of parameters in large collections of tissue or cell samples.  

We currently have an inventory of ~220 validated antibodies that cover multiple total proteins and phosphoproteins in the following protein pathways or functional protein groups: epithelial-mesenchymal transition (EMT), stem cells, apoptosis, DNA damage, proliferation and cell cycle, growth factor receptors, cytokines/STATs, nuclear receptors/transcriptional regulatory proteins, autophagy and metabolomics enzymes. We continually work together with Baylor investigators to build RPPA assays for new protein pathways of interest and to validate the required antibodies. We are also working to be able to do analysis with various types of samples including cell/tissue lysates, macro-dissected tumor samples, laser capture micro-dissected (LCM) samples, small numbers of isolated stem cells or other rare cell types, and body fluids such as serum and urine. A novel application we have developed is to analyze isolated protein complexes. We have developed in-house normalization and statistical analyses algorithms using the R statistical software. View the R Project website.

Antibody Validation

Validation criteria include a single or predominant band of the correct size by immunoblot assay of appropriate control lysates plus proper performance and response by RPPA with positive and negative controls and calibrator lysates. Antibodies are validated in house or by collaboration with other RPPA cores and validation data can be provided upon request. 

Infrastructure

Major Equipment:

Aushon 2470 Protein Arrayer
DAKO Link 48 Autostainer
Axon Array Scanner 4200AL
TissueLyzer
NanoDrop
SpectraMax 340PC(384) plate reader

Personnel:
Core Director: Shixia Huang, Ph.D.
Lab Research Staff
Fuli Jia, M.S.
Danli Wu, Ph.D.

Bioinformaticians/Statisticians:
Cristian Coarfa, Ph.D.
Kimal Rajapakshe, Ph.D.

Location:
ABBR Building, Main BCM Campus, 5th Floor R551C

RPPA Services

We provide the following levels of services:
1. consultation; 2. technology platform services; and 3. primary data analyses

Charge rates in the table are for the above RPPA services.

Additional higher level statistical and data analyses services, please inquire for charge rates with Dr. Cristian Coarfa.

1. Consultation:

1) Consultation on experimental design
2) Consultation on sample preparation.
3) When data is ready, review data with PI, help with analysis strategies and assist with results interpretation.

When PI is interested in RPPA assays, an initial meeting will be set up between core director and PI and associates to discuss the project design. The discussion is facilitated by submission of two forms: project information form and the RFP form.

Once the project design is finalized, core director or laboratory staff will meet with a researcher to go through the critical steps of sample preparation, protein concentration measurement, and preparation of RPPA-ready samples.

When data is ready, a meeting is set up to review data with PI, help with analysis strategies and assist with results interpretation.

2. Technology Platform Services: (RPPA laboratory services)

1) Prepare and provide lysis buffer and protocols for cell/tissue lysis.
2) Active communication with researcher to ensure sample quality.
4) Provide test printing service option for rare samples.
5) Provide lysate preparation service option for tissue samples or cell lines samples for laboratories needing help for such services.
6) Go through sample submission form and concentration to ensure quality.
7) Process lysates and transfer to 384 well plates.
8) Program protein arrayer, and print and array slides.
9) Antibody labeling of printed slides with DAKO autostainer.
10) Negative control slides by omitting primary antibody.
11) Slides for total protein by Sypro Ruby Staining
12) Provide a wide-range of control lysates and calibrators on each slide (see detailed protocols).

3. Primary Data Analyses:

1) Fluorescence scanning of antibody reacted slides: each slide is scanned with 2-5 PMT settings depending on the signal intensity of the particular antibody. We strive to obtain an optimal setting for each set of samples from different laboratories. We routinely generate 700-1000 images for >200 antibodies, negative controls, and total protein stained slides.
2) Image Analyses of each scanned slides by Axon GenePix Software 7.0.
(Automatic spot finding, manual spot adjustment, and background subtraction).
3) Spot quality control: spot-by-spot quality inspection by experienced technical personnel.
4) Normalization by subtraction of negative control signal/spot/slide.
5) Normalization by total protein signal.
6) Generate normalized images.
7) QC normalization process by statistician & laboratory personnel.
8) Quality control assessment on controls & samples by statistician and laboratory personnel.
9) Generate data for each PI using the most appropriate setting for the specific set of samples
10) Provide primary data to investigator as Excel spreadsheets with graphical analysis of quality control lysates and calibrators. Each PI receives one excel file for their samples with 4 worksheets (legend, normalized data, quality index, and raw data) for rabbit/goat antibodies or 7 worksheets (legend, normalized data, quality index, and raw data, mouse normalized data, mouse quality index, and mouse raw data) for mouse antibodies. This one excel file contains data extracted from the 700-1000 images describes in #1 above.
11) Review data with PI, help with analysis strategies and assist with results interpretation.

Additional Higher Level Data Analyses

Please contact Dr. Cristian Coarfa.

  • Project-orientated analysis such as testing hypotheses using appropriate statistical methods
  • Preparing tables and graphs for presentations and publications
  • Generate bar graphs and/or heat maps for each antibody
  • Integrate RPPA data with gene expression or other data sets
  • Pathway mapping
  • Additional data-relevant consultations and defining additional experiments

Validation of RPPA Results

RPPA results require validation by an independent assay that typically is an immunoblot assay.  The Core can provide investigators with small aliquots of the same antibodies used in RPPA for immunoblots to be performed.

References (Core Supported Publications)

Dong S, Jia C, Zhang S, Fan G, Li Y, Shan P, Sun L, Xiao W, Li L, Zheng Y, Liu J, Wei H, Hu C, Zhang W, Chin YE, Zhai Q, Li Q, Liu J, Jia F, Mo Q, Edwards DP, Huang S, Chan L, O'Malley BW, Li X, Wang C. The REGγ proteasome regulates hepatic lipid metabolism through inhibition of autophagy. Cell Metab. 2013 Sep 3;18(3):380-91. doi: 10.1016/j.cmet.2013.08.012. PubMed PMID: 24011073; PubMed Central PMCID: PMC3813599.

Chang CH, Zhang M, Rajapakshe K, Coarfa C, Edwards D, Huang S, Rosen JM. Mammary Stem Cells and Tumor-Initiating Cells Are More Resistant to Apoptosis and Exhibit Increased DNA Repair Activity in Response to DNA Damage. Stem Cell Reports. 2015 Sep 8;5(3):378-91. doi: 10.1016/j.stemcr.2015.07.009. Epub 2015 Aug 20. PubMed PMID: 26300228; PubMed Central PMCID: PMC4618454.

Holdman XB, Welte T, Rajapakshe K, Pond A, Coarfa C, Mo Q, Huang S, Hilsenbeck SG, Edwards DP, Zhang X, Rosen JM. Upregulation of EGFR signaling is correlated with tumor stroma remodeling and tumor recurrence in FGFR1-driven breast cancer.  Breast Cancer Res. 2015 Nov 18;17:141. doi: 10.1186/s13058-015-0649-1. PubMed PMID: 26581390; PubMed Central PMCID: PMC4652386.

Methods and Reviews: 

Creighton CJ, Huang S. Reverse phase protein arrays in signaling pathways: a data integration perspective. Drug Des Devel Ther. 2015 Jul 7;9:3519-27. Review. PubMed PMID: 26185419; PubMed Central PMCID: PMC4500628.