Reference Genes Selection for Transcriptional Profiling in Blood of HD Patients and R6/2 Mice

Abstract

Background: Huntington's disease is a neurodegenerative disorder characterized by transcriptional alterations both in central and peripheral tissues. Therefore, the identification of a transcriptional signature in an accessible tissue can meaningfully complement current efforts in clinical biomarker development. Gene expression normalization represents an essential step in transcriptional signatures identification, and since many reference genes show altered expressions in several pathologies, the definition of stable genes in the desired tissue is required to allow correct result interpretations. Objective: The present work aimed at identifying a set of suitable reference genes for expression normalization in blood of HD patients and R6/2 mice. Methods: By crossing literature investigation and analysis of microarrays performed on blood of HD patients and healthy subjects, a set of genes was selected and tested by RT-qPCR. Employment of statistical algorithms allowed the identification of the most stable genes in human samples that were than confirmed in R6/2. Results: PPIB, PGK1, ACTB and YWHAZ represent the best possible genes combination, useful to normalize blood transcriptional analysis. To link clinical and preclinical studies, the identified genes were investigated also in blood of R6/2 and wild type mice, confirming that Ppib, Actb and Ywhaz were appropriate for expression normalization. Selected references were subsequently applied to evaluate expression of genes known to be involved in Huntington's pathological progression. Conclusions: This work highlights the importance for correct data normalization to avoid misinterpretation of results, while providing a suitable method to support quantitative gene expression analysis in preclinical and clinical investigations.

MicroRNAs as Diagnostic and Prognostic Biomarkers in Ischemic Stroke—A Comprehensive Review and Bioinformatic Analysis

“…Our results support the importance of HMGB in IS. The role of YWHAZ gene was shown in several neurodegenerative diseases such as, Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis [89,90,91]. As we found in our bioinformatic analysis a similar study showed the importance of YWHAZ gene in CE [92].…”

MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation

“…qRT–PCR was performed using the Power SYBR Green RNA-to-CT 1-Step Kit (Life Technologies) and a 7900 HT Sequence Detection System with SDS 2.2 software (Applied Biosystems). Gene expression was normalized to PGK1 expression because PGK1 was not differentially expressed in the microarray data and was previously identified as a reference gene for gene expression studies 51 52 53 . A gene was considered to validate for a given comparison if it met the following two criteria: (i) the gene was differentially expressed in both microarray (B–H adjusted eBayes P value <0.05) and qRT–PCR data (Student's t -test P value <0.05), and (ii) the gene had the same direction of expression change in both microarray and qRT–PCR data.…”

Whole gene expression profile in blood reveals multiple pathways deregulation in R6/2 mouse model

“…R6/2 is one of the first HD transgenic mouse model created, expressing only the N-terminal fragment of HTT (exon 1) and it is characterized by short survival and development of pathological features mimicking human stages of disease [ 13 , 14 ]. For this reason, transcriptional and behavioral alterations have been intensely studied in R6/2, which has being commonly employed not only in pre-clinical drug testing but also in peripheral investigations, often realized in parallel with HD patients [ 15 , 16 ]. Stemming from those observations, although HD is mainly a CNS disorder, ubiquitous expression of both normal and mut-HTT in the whole body [ 17 ] has moved the attention toward peripheral dysfunctions, increasing the knowledge on HD etiology and other disease manifestations, and aiding the finding of new biomarkers [ 18 , 19 ], as evidenced by the flourishing number of transcriptional and proteomic studies conducted in HD blood [ 16 , 20 - 24 ].…”