RNA-sequencing (RNA-seq) is poised to play a major role in the management of kidney transplant patients. It is very desirable to develop a set of genes with housekeeping function (GHF) to facilitate quantitative comparison of gene expression across samples. The use of single genes such as 18s rRNA and vimentin is inappropriate as these can be over-expressed in acute rejection.

30 biopsies were studied from patients with stable function, acute rejection, interstitial nephritis, interstitial fibrosis or BK nephritis. RNA-seq was performed and data normalized using 9 methods, including Total Counts, library size, Trimmed Median of Means, Upper Quartiles, median, Quantiles, DESeq, TPKM and RPKM. Transcripts with coefficient of variation <2% were considered suitable candidates to be designated as GHF set.

A total of 47611 transcripts common to all samples was obtained. Bias and variance for different normalization methods was the highest for the RPKM method, and the least for the Quantile method. GHF constituted 0.60% of genes expressed in diseased allograft derived renal transcriptomic signatures available in Gene Expression Omnibus (GEO) Database. A set of 42 transcripts was confirmed by all normalization methods, and was therefore defined as GHF. Cell morphology, cellular assembly and organization, cellular function and maintenance, cell cycle and gene expression were identified as the top 5 molecular and cellular functions involved in GHF set. This gene set had only 2 genes in common with the Affymetrix U133A Normal Control Probe set, which is derived from 100 human genes and is commonly used on the DNA microarray platform. However, 2D line plots showed comparable gene expression profiles for both gene sets when applied to RNAseq platform using data obtained from formalin fixed renal allograft biopsies

In conclusion, we have identified a set of 42 GHF in renal allograft tissues to facilitate development of robust molecular diagnostic methods applicable to formalin fixed renal allograft biopsies. Data normalization using all 42 genes will prevent the loss of power that results from utilization of genes such as GAPDH and beta-actin which can be altered by transplant related disease states.

CITATION INFORMATION: Wang Z., Pan L., Lyu Z., Zeng G., Randhawa P. Genes with Housekeeping Function (GHF) in Renal Allograft Tissue: An RNA-Seq Analysis Am J Transplant. 2017;17 (suppl 3).

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