A detailed and radioisotope-free protocol for electrophoretic mobility shift assay (EMSA)

To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. This protocol is to provide a detailed procedure for a safe EMSA assay (without using any radioisotope).

Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNA

Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodology: EMSA analysis showed that PPB depended on siRNA and plasma concentration. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiologically relevant siRNA concentrations. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metabolism and excretion and advanced development of RNA interference therapeutics.

The rs75862629 minor allele in the endoplasmic reticulum aminopeptidases intergenic region affects human leucocyte antigen B27 expression and protects from ankylosing spondylitis in Sardinia

Objectives HLA-B27 and the endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 genes are predisposing factors for AS. A single nucleotide polymorphism (SNP) in the ERAP2 promoter (rs75862629) coordinates the transcription of both ERAP genes. We investigated whether this SNP associates with AS and whether it affects the expression of the two major HLA-B27 alleles present in Sardinia, the AS-associated B*2705 and the non-AS-associated B*2709. Methods Four SNPs in the ERAP region were genotyped in HLA-B*2705-positive patients with AS (n = 145), B27-positive healthy subjects (n = 126) and B27-negative controls (n = 250) and the allele and haplotype frequencies were derived. The expression of ERAP1 and ERAP2 mRNAs in 36 HLA-B27-positive B lymphoblastoid cell lines was measured by quantitative PCR. An electrophoretic mobility shift assay was performed to search for a nuclear factor binding the DNA sequence encompassing rs75862629. The expression of HLA-B27 molecules related to the SNP at rs75862629 was determined by flow cytometry. Results The minor allele G at rs75862629 was found significantly increased in B27 healthy individuals, both B*2705 and B*2709, compared with B*2705-positive patients with AS and B27-negative controls. The electrophoretic mobility shift assay indicated the lack of binding of a transcription factor as the cause of the observed reduction in the ERAP2 concomitant with a higher ERAP1 expression. Of note, this occurs with a different cell surface expression of the HLA-B*2705 and HLA-B*2709 molecules. Conclusion SNP rs75862629, by modulating simultaneously the expression of ERAP1 and ERAP2, provides protection from AS in HLA-B27-positive subjects in Sardinia. This has a functional impact on HLA-B27 expression and likely on disease onset.