TY - JOUR
T1 - Spatial normalization of reverse phase protein array data
AU - Kaushik, Poorvi
AU - Molinelli, Evan J.
AU - Miller, Martin L.
AU - Wang, Weiqing
AU - Korkut, Anil
AU - Liu, Wenbin
AU - Ju, Zhenlin
AU - Lu, Yiling
AU - Mills, Gordon
AU - Sander, Chris
N1 - Publisher Copyright:
© 2014 Kaushik et al.
PY - 2014/12/12
Y1 - 2014/12/12
N2 - Reverse phase protein arrays (RPPA) are an efficient, high-throughput, cost-effective method for the quantification of specific proteins in complex biological samples. The quality of RPPA data may be affected by various sources of error. One of these, spatial variation, is caused by uneven exposure of different parts of an RPPA slide to the reagents used in protein detection. We present a method for the determination and correction of systematic spatial variation in RPPA slides using positive control spots printed on each slide. The method uses a simple bi-linear interpolation technique to obtain a surface representing the spatial variation occurring across the dimensions of a slide. This surface is used to calculate correction factors that can normalize the relative protein concentrations of the samples on each slide. The adoption of the method results in increased agreement between technical and biological replicates of various tumor and cell-line derived samples. Further, in data from a study of the melanoma cell-line SKMEL-133, several slides that had previously been rejected because they had a coefficient of variation (CV) greater than 15%, are rescued by reduction of CV below this threshold in each case. The method is implemented in the R statistical programing language. It is compatible with MicroVigene and SuperCurve, packages commonly used in RPPA data analysis. The method is made available, along with suggestions for implementation, at http://bitbucket.org/rppa-preprocess/rppa-preprocess/src.
AB - Reverse phase protein arrays (RPPA) are an efficient, high-throughput, cost-effective method for the quantification of specific proteins in complex biological samples. The quality of RPPA data may be affected by various sources of error. One of these, spatial variation, is caused by uneven exposure of different parts of an RPPA slide to the reagents used in protein detection. We present a method for the determination and correction of systematic spatial variation in RPPA slides using positive control spots printed on each slide. The method uses a simple bi-linear interpolation technique to obtain a surface representing the spatial variation occurring across the dimensions of a slide. This surface is used to calculate correction factors that can normalize the relative protein concentrations of the samples on each slide. The adoption of the method results in increased agreement between technical and biological replicates of various tumor and cell-line derived samples. Further, in data from a study of the melanoma cell-line SKMEL-133, several slides that had previously been rejected because they had a coefficient of variation (CV) greater than 15%, are rescued by reduction of CV below this threshold in each case. The method is implemented in the R statistical programing language. It is compatible with MicroVigene and SuperCurve, packages commonly used in RPPA data analysis. The method is made available, along with suggestions for implementation, at http://bitbucket.org/rppa-preprocess/rppa-preprocess/src.
UR - http://www.scopus.com/inward/record.url?scp=84917706891&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84917706891&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0097213
DO - 10.1371/journal.pone.0097213
M3 - Article
C2 - 25501559
AN - SCOPUS:84917706891
SN - 1932-6203
VL - 9
JO - PloS one
JF - PloS one
IS - 12
M1 - e97213
ER -