QRT-PCR Gene Expression Analysis in Monocytes from Normal and Alpha 1-Antitrypsin Deficient Individuals Using Total RNA or SideStep Cell Lysates.

56 Background: Gene expression profiling has been shown to be effective in analyzing postoperative tumor samples in various cancers. However, in analyzing small specimens such as core biopsies, the limited amount of available material makes multi-gene analyses difficult or impossible. Microarray-based analyses also provide limited dynamic range. We describe the development of targeted RNA-sequencing methodology which combines the power of a universal RNA amplification with NGS for an ultra-deep expression analysis of multiple target genes, enabling <100 ng of sample input for multi-gene analysis in a single tube format. Methods: The gene expression patterns of triple-negative breast cancer FFPE samples were analyzed using a 96-gene breast cancer biomarker panel across three different platforms: Affymetrix Human Gene ST 1.0 microarrays, a pre-developed OncoScore qRT-PCR panel, and targeted RNA-seq. For targeted RNA-seq analysis, the 96-gene panel was amplified using a universal, single-tube “XP-PCR” amplification strategy followed by sequence analysis using the Ion-Torrent Personal Genome Machine. Results: Targeted RNA-seq provided the most sensitivity in terms of detection rates with <100 ng FFPE RNA input and provides unlimited dynamic range with increased sequencing depth. Expression ratio compression issues typically associated with a high number of pre-amplification cycles in standard multiplex-primed methods were not observed here. Low expressing genes, undetectable by qRT-PCR analysis from 1,000 ng input FFPE RNA, were detected and eligible for expression analysis with a significant number of sequencing reads. Alternative transcription/splicing analysis is also possible from sequence analysis of the target transcripts using targeted RNA-seq. Conclusions: By combining universally primed pre-amplification and NGS in multi-gene expression analysis, targeted RNA-seq provides the most sensitive gene expression analysis methodology.

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Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

10.1101/2020.08.12.247601 ◽

2020 ◽

Author(s):

Liz M. Florez ◽

Reiny W. A. Scheper ◽

Brent M. Fisher ◽

Paul W. Sutherland ◽

Matthew D. Templeton ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Time Course ◽

Gene Expression Analysis ◽

Virulence Genes ◽

Functional Characterization ◽

Post Inoculation ◽

In Planta ◽

Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

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