Reference Genes for Expression Studies in Human CD8 + Naïve and Effector Memory T Cells under Resting and Activating Conditions

Abstract: Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used for mRNA quantification. To accurately measure changing gene transcript levels under different experimental conditions, the use of appropriate reference gene transcripts is instrumental. In T cell immunology, suitable reference genes have been reported for bulk CD4+ and CD8+ T cells. However, many CD4+ and CD8+ T cell subsets have been described in the past. Although they respond differently to given activation stimuli, proper validation of suitable reference genes in these subsets is lacking. In this study, we evaluated twelve commonly used reference gene products in human naïve (NV) and effector memory (EM) CD8+ T cells under non-activated and activated (2 h, 10 h and 20 h) conditions. We used five different statistical approaches for data analysis. Our results show that a number of widely used reference transcripts become differentially expressed under activating conditions. Using them as references markedly alters results as exemplified with IFNG mRNA expression. The only candidate reference gene products that remained stable during the activation process were 18S rRNA and SDHA mRNA, encouraging their usage as reference gene products for RT-qPCR experiments, when quantifying mRNA levels in human NV and EM CD8+ T cells

A natural experiment study: Low-profile double plating versus single plating techniques in midshaft clavicle fractures-Study protocol.

BackgroundSingle plate osteosynthesis is commonly employed when performing surgical stabilization of midshaft clavicle fractures. In recent years, a smaller structural low-profile double plating technique has been described as a possible solution for the high removal rates associated with single plating. A previous meta-analysis has demonstrated that low-profile double plating attains the same healing rates as single plating without a higher chance of fracture-related infections. This meta-analysis, however, was based on relatively small studies. Therefore, a multicentre prospective natural experiment was designed using natural variation in treatment regimens and geographical location of the trauma as treatment allocation mechanism to compare both treatments on a larger scale. This manuscript describes its protocol.Material & methodsPatients (≥16 years) with primary midshaft clavicle fractures that are eligible for operative treatment will be included. Treatment allocation will be determined by the geographical location of the accident and local hospital providing treatment. In two centres, single plating is the treatment of choice for these patients. In two others, low-profile double plating has become the standard treatment. For the low-profile double plating group, one superiorly positioned VariAx 2.0mm and one anterior VariAx 2.4mm or 2.7mm plate will be used. For the single plating group, the standard locally available implant will be used. A total of 336 patients will be included. The primary outcome of interest is re-intervention. Secondary outcomes include complications, operative time, length of incision, functional scores (DASH, EQ-5D-DL, VAS-Pain/Satisfaction) and cost-effectiveness.DiscussionThis study will determine whether low-profile double plating has significant clinical and cost-effective benefits over single plating techniques in midshaft clavicle fractures. The study will also give insight in the performance of a natural experiment study design for orthopedic trauma research.Trial registrationThis study has been registered on ClincialTrials.gov, identifier NCT05579873