An RNA spiking method demonstrates that 18S rRNA is regulated by progesterone in the mouse uterus

Abstract

Identifying suitable housekeeping genes for quantitative RT-PCR in the uterus is problematic, as this tissue undergoes significant structural and functional alterations during the oestrous cycle and pregnancy in response to circulating hormones. The suitability of 18S rRNA as a housekeeping gene in mouse uterus was investigated by introducing an 'RNA spike' standard into the reverse transcription reaction. 18S rRNA levels increased by Day 4 of pregnancy and after progesterone administration in ovariectomized mice. We conclude that 18S rRNA is not a suitable housekeeping gene for quantitative RT-PCR analysis in progesterone-responsive tissues, and the RNA spiking method provides a suitable alternative.

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus

“…Each cDNA master-mix reaction contained an equal amount of in vitro-transcribed RNA spike and a 300-bp region of the T7 promoter region of the pBluescript phagemid ). This spike controls for the efficiency of the cDNA reaction (Craythorn et al 2009;Winnall et al 2009). Following cDNA synthesis, samples were diluted 1 : 10 in nuclease-free sterile water.…”

“…Messenger RNA from PND0 FSKO mice, ovarian tissue from adult WT mice and human myometrium were included as internal controls. The RNA spike was used to normalise the data (Craythorn et al 2009;Winnall et al 2009) and was detected at the same level in each tissue (data not shown). Utilisation of spike was particularly important for investigations of the oestrous-cycle stage as housekeeping genes, such as 18S, are influenced by steroid hormones (Craythorn et al 2009).…”

“…The RNA spike was used to normalise the data (Craythorn et al 2009;Winnall et al 2009) and was detected at the same level in each tissue (data not shown). Utilisation of spike was particularly important for investigations of the oestrous-cycle stage as housekeeping genes, such as 18S, are influenced by steroid hormones (Craythorn et al 2009). All data were analysed by relative quantification to the RNA spike, using the method described by Pfaffl (2001).…”

Acute regulation of activin A and its binding protein, follistatin, in serum and tissues following lipopolysaccharide treatment of adult male mice

“…Tissue samples were processed and assessed for mRNA expression levels using established methods, which have been described in detail previously (6,41). All tissues for mRNA measurements were stored at [minus]80°C.…”

“…For each tissue cDNA, a nonreverse-transcribed control was produced to act as an internal negative control. A phagemid-derived RNA "spike" was added to each reaction as an external standard control, as previously described (6,41).…”