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Effect of different reverse transcription aproaches in PRU P 3 transcripts semiquantitative amplification

Abstract:

Reverse transcriptase transcribes the cDNA based on its previous extraction and standardization. Reverse transcription step is considered to be critical in the workflow of quantification of transcribed genes. The aim of the study was to extract total RNA by different methods and to analyse the results of the subsequent reverse transcription reaction when different commercial RT kits were used to process RNA extracted from pulp of matured peach fruit. Mature peach pulp was used in the study. The fruit of variety Vistarich was collected in summer 2017 in the orchard of Dvory nad Titavou. Two RNA extraction methods, TRIzol® Reagent and GeneJET Plant RNA Purification Kit, were tested in to determine the suitable method for peach fruit RNA extraction. Three different cDNA reagent sets were used to transcribe 115 ng/500 ng total RNA or 11 ng/115 ng, respectively. Both variants of the primers, random hexamers as well as oligo (dT) 18, were used to anneal the target mRNA of Pru p 3 allergen following the manufacturer instructions. No specific effect was obtained in the case of peach fruit when using ethanol-extracted tissue treatment and the effect of the used extraction method was more significant. The A260/230 ratios were similar for three from four tested methods. In the case of these three methods, the A260/A230 ratios for all the extracted samples were higher than 1.9 which indicates high purity without contamination by polyphenols and polysaccharides. The specificity of obtained amplicons was proved by restriction cleavage using Tse I restriction endonuclease. This provided the cleavage of the 179 bp long product in all amplicons. Working with mature fruit meet a specific situation in the field of RNA extraction and subsequently all the downstream applications. That is, why choosing the most fitting methods and kits is a crucial step. Here, the method for the semi-quantitative analysis of the Pru p 3 allergen expressions was set up in the way that will be directly applicable for Pru p 3 expression analyses.