18S rRNAis a reliable normalisation gene for real time PCR based on influenza virus infected cells

Abstract

BackgroundOne requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes.ResultsThe relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells.ConclusionsBased on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.

Selection of reliable reference genes for RT-qPCR studies in Octopus vulgaris paralarvae during development and immune-stimulation

“…The 18S is a classical gene used as internal control for RT-qPCR with controversial results. The expression of 18S was found unstable in Crassosstrea gigas development (Du et al 2013), however, the same gene remained stable in cells infected by virus (Kuchipudi et al 2012). In this study, the suitability of 18S as an appropriate reference gene for normalizing data of octopus paralarvae development is supported by its stability tested by different software ( (Zhang et al 2014); and in muscle, gill and spleen of red drum, Sciaenops ocellatus, infected with Edwardsiella tarda (Sun and Hu 2015).…”

Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides

“…In order to obtain a suitable reference gene or group of genes for expression analysis in the dimorphic process in M. circinelloides, we selected seven different genes that have been proposed previously as reference genes for qRT-PCR in different organisms and the corresponding hydrolysis probes were designed: as follows: (1) vma-1, encoding the catalytic subunit of the vacuolar ATPase [17]; (2) tfc-1, encoding a subunit of the transcription factor TFIIIC from RNA polymerase III [18]; (3) ef-1, encoding the translation elongation factor 1 [19]; (4) tfb-4, encoding the subunit of the Transcription factor II human (TFIIH) complex, involved in transcription initiation [20]; (5) e1, for the ubiquitin-activating enzyme [21]; (6) the 18S, which encodes the 18S ribosomal RNA (rRNA) [22] and (7) the 28S gene, which encodes the 28S rRNA [23]. (Table S1, Fig.…”

Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

“…Because iron chelators were shown to result in accumulation of cells at G 1 phase of the cell cycle, we examined the effects of PPY-based iron chelators on the expression levels of CDK2-associated cyclins and the CDK9-dependent genes for HLA and IB-␣. We used 18S RNA as a normalization housekeeping control, as it was shown to be the most reliable housekeeping control among several tested, including ACTB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (37). Treatment with PPYaT or PPYeT induced expression of cyclin A and cyclin E (Fig.…”