Extraction of RNA from formalin‐fixed, paraffin‐embedded equine placenta

Abstract

Contents Archived formalin‐fixed, paraffin‐embedded (FFPE) samples represent a valuable resource for the determination of gene expression for physio/pathological conditions. In the present study, we validated a protocol for the extraction of RNA from FFPE samples collected from healthy and diseased equine placenta. The quality and quantity of the extracted RNA from the FFPE and matching RNAlater™‐preserved samples and expression levels of common housekeeping genes and reference microRNAs were evaluated. Precision of the expression data was evaluated by comparing relative expression of CYP19A1 and HSD3B1 in FFPE and RNAlater™ samples. The median RNA concentration recovered from FFPE samples was 316.8 ng/mm3 of tissue (ranging between 61.6 and 917.4 ng/mm3), average RNA integrity number was 2.3 ± 0.9 (mean ± standard deviation), and 84% of samples had RNA fragments longer than 200 nucleotides (DV200). RNA concentrations and CT values for GAPDH, ACTB, miR‐8908a and miR‐369 in FFPE samples were significantly correlated (r = −0.8, −0.7, −0.4 and −0.4, respectively; p < 0.001). Expression pattern of normalized CYP19A1 and HSD3B1 in paired FFPE and RNAlater™ samples was significantly correlated (r = 0.97 for CYP19A1 and HSD3B1; p < 0.001). This study demonstrates that RNA can be extracted from FFPE equine placental tissue and used for downstream transcriptomic analysis. Similar RNA expression patterns were obtained using RNAlater™ and FFPE tissue samples.

Estrogens Regulate Placental Angiogenesis in Horses

“…The PCR efficiency was calculated using LinRegPCR (version 2012.0) (http://www.hartfaalcentrum.nl; accessed in 2 March 2019). The relative expression of each gene (∆CT) was calculated where ∆CT = (CT values of the gene of interest-geometric mean CT values of ACTB and GAPDH) [51,52].…”

Paternally expressed retrotransposon Gag-like 1 gene, RTL1, is one of the crucial elements for placental angiogenesis in horses

“…Melt curve analysis was performed to check for non-specific amplifications along with the inclusion of non-template controls. Reactions were performed in duplicate and GAPDH, and ACTB were used as housekeeping genes [76,77]. PCR efficiencies were calculated using LinRegPCR (version 2012.0) (http://www.hartfaalcentrum.nl) to ensure that all primers resulted in PCR efficiencies between 1.8 and 2.1.…”

“…We previously tested and confirmed the efficiency of RNA isolation from FFPE equine placental tissues [76]. Briefly, isolation of total RNA from FFPE specimens was performed using the FFPE RNA Purification Kit (#25300; Norgen Biotek Corp, Ontario, Canada).…”