Abundance, efficiency, and stability of reference transcript expression in a seasonal rodent: The Siberian hamster

Abstract

Quantitative PCR (qPCR) is a common molecular tool to analyse the expression of transcripts in non-traditional animal models. Most animals experience tissue-specific seasonal changes in cell structure, growth, and cellular function. As a consequence, the choice of reference or ‘house-keeping’ genes is essential to standardize expression levels of target transcripts of interest for qPCR analyses. This study aimed to determine the abundance, efficiency and stability of several reference genes commonly used for normalisation of qPCR analyses in a model of seasonal biology: the Siberian hamster (Phodopus sungorus). Liver, brown-adipose tissue (BAT), white adipose tissue (WAT), testes, spleen, kidney, the hypothalamic arcuate nucleus, and the pituitary gland from either long or short photoperiod Siberian hamsters were dissected to test tissue-specific and photoperiod effects on reference transcripts. qPCR was conducted for common reference genes including 18s ribosomal RNA (18s), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), hypoxanthine-guanine phosphoribosyltransferase (Hprt), and actin-β (Act). Cycling time (Ct), efficiency (E) and replicate variation of Ct and E measured by percent coefficient of variance (CV%) was determined using PCR miner. Measures of stability were assessed using a combined approach of NormFinder and BestKeeper. 18s and Act did not vary in Ct across photoperiod conditions. Splenic, WAT and BAT Gapdh Ct was higher in long compared to short photoperiod. Splenic Hprt Ct was higher in long photoperiods. There was no significant effect of photoperiod, tissue or interaction on measures of efficiency, Ct CV%, or efficiency CV%. NormFinder and BestKeeper confirmed that 18s, Gapdh and Hprt were highly stable, while Act showed low stability. These findings suggest that 18s and Hprt show the most reliable stability, efficiency, and abundance across the tissues. Overall, the study provides a comprehensive and standardised approach to assess multiple reference genes in the Siberian hamster and help to inform molecular assays used in studies of photoperiodism.

Prolactin Mediates Long-Term, Seasonal Rheostatic Regulation of Body Mass in Female Mammals

“…As an endocrine factor, prolactin can be a powerful output signal to multiple physiological “effector” tissues. Prolactin receptors are distributed widely across many tissues, including those essential for reproduction (ie, uterine, ovary, and testes), osmoregulation (ie, kidney), and energy balance (eg, adipose, liver) ( 39 ). Prolactin receptors belong to the cytokine receptor family and ligand binding leads to tyrosine phosphorylation of several cellular proteins via the Janus kinase (JAK)—signal transducers and activators of transcription 5 (STAT) pathway ( 42 ).…”

“…To examine molecular substrates that underlie programmed rheostatic control of energy balance, primer pairs were used that targeted Sst , Cart , Npy , Agrp , Dio3 , Th , Tshβ , Prl , Prlr, and Gh (Supplementary Table S1 ( 38 )). Reference transcripts that were selected based on a previous publication ( 39 ) that demonstrated high stability included 18 seconds , Gapdh and Hprt (Supplementary Table S1 ( 38 )). Reactions were carried out in duplicate and run with non-template negative controls in an Mx3000P qPCR system (Agilent) using the following thermal cycling conditions: (i) initial denaturation at 95 °C for 7.5 minutes followed by 40-45 cycles of (ii) denaturation at 95 °C for 30 seconds, (iii) annealing at temperature specified for each respective gene in Supplementary Table S1 ( 38 ) for 30 seconds, and (iv) extension at 72 °C for 30 seconds.…”