Extraction of RNA from fermented milk products for in situ gene expression analysis

e14731 Background: Ductal carcinoma in situ (DCIS) display favorable outcome while little is known about the factors associated with invasive recurrence. To identify better prognostic biomarkers we performed gene expression analysis followed by immunohistochemistry (IHC) staining validation. Methods: Differential gene expression analysis of 29 pure DCIS patients was performed using nanostring platform. RNA was extracted from paraffin blocks from age/size matched 11 recurrence-free and 18 invasive-recurrence cases (disease free interval > 5 years). Gene annotation enrichment analysis was done for differentially expressed genes (DEG) using DAVID. Eighty-two pure DCIS cases were selected for external validation by IHC staining. Allred score cutoff 1 was used for survival analysis. Results: Ninety-nine differentially expressed genes were found statistically significant (p-value < 0.05). Androgen receptor (AR) gene, which encodes a transcription factor AR, has recently been highlighted as a favorable prognostic marker and a therapeutic target in invasive tumor (fold change = - 1.35, p < 0.001). AR protein expression was externally validated by IHC staining of 82 pure DCIS cases (24 invasive-recurrence versus 58 recurrence-free). Similar to gene expression analysis result, patients with invasive recurrence showed lower AR staining score than recurrence-free patients (p = 0.007). Cox regression analysis showed lower AR level as an independent risk factor of long-term invasive recurrence (HR 7.43, 95%CI 1.50 – 36.62). Gene enrichment analysis revealed enrichment of kinase pathway and cell cycle pathway in recurred cases (Enrichment Score = 2.43, 2.41 respectively). Conclusions: DEG pattern was observed among pure DCIS cases. AR may serve as a prognostic biomarker and targeting kinase, cell proliferation may be effective for higher risk DCIS patients.

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Minimally invasive, pressure probe based sampling allows for in-situ gene expression analyses in plant cells

10.1101/768978 ◽

2019 ◽

Author(s):

Hiroshi Wada ◽

Simone D. Castellarin ◽

Mark A. Matthews ◽

Kenneth A. Shackel ◽

Gregory A. Gambetta

Keyword(s):

Gene Expression ◽

Minimally Invasive ◽

Single Cell ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Plant Cells ◽

Pressure Probe ◽

Plant Tissues ◽

Gene Expression Analyses

AbstractBackgroundGene expression analyses are conducted using multiple approaches and increasingly research has been focused on assessing gene expression at the level of a tissue or even single-cells. To date, methods to assess gene expression at the single-cell in plant tissues have been semi-quantitative, require tissue disruption, and/or involve laborious, possibly artifact-inducing manipulation. In this work, we used grape berries (Vitis vinifera L. Zinfandel) as a model in order to examine the validity and reproducibility of an in-situ gene expression analysis method combining a cell pressure probe (CPP) with quantitative PCR (qPCR).ResultsWe developed a method to directly assess gene expression levels via qPCR from cellular fluids sampled in-situ with a CPP. Cellular fluids, with volumes in the picoliter range, were collected from intact berries with a CPP at various depths across skin and mesocarp tissues. The expression of a key anthocyanin biosynthetic gene, UDP-glucose: flavonoid 3-O-glucosyltransferase (VviUFGT), was analyzed as a test case since its expression is restricted to cells producing anthocyanins in grape berry skins during ripening. The method identifies samples contaminated with significant levels of genomic DNA by amplifying a region of VviUFGT that spans an intron. Therefore false positives were discarded which occurred in 28% of the samples tested. Shallow probing of skin cells showed high VviUFGT expression as expected while deeper probing of mesocarp cells resulted in no VviUFGT expression.ConclusionsThe clear correspondence of VviUFGT expression to the targeted cell samples suggests that the in-situ gene expression analysis using a CPP is reliable and does not result in contamination as the probe moves through tissues. This method can be paired to single-cell transcriptomic analyses in the future. We conclude that this technique represents a minimally invasive method of sampling plant cells in-situ which creates an opportunity for the analysis of cellular level, spatiotemporal responses in heterogeneous plant tissues.

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