Evaluation of different RNA extraction methods for high-quality total RNA and mRNA from Erwinia amylovora in planta

Abstract

Obtaining a sufficient quantity of high-quality, intact RNA is the first crucial step in its study by RNA sequencing on next-generation sequencing platforms or quantitative PCR. Different RNA extraction methods or commercial kits vary in yield and in the quality and integrity of the RNA obtained, which may affect the results of downstream applications. Often, these factors depend on the organism under study and nature of the sample. Therefore, the selection of an appropriate RNA isolation method is critical. In this study, we present the results of an evaluation of three different commercial kits for the isolation of total RNA from Erwinia amylovora in apple tissue as well as the usefulness of different kinds of Deoxyribonuclease I for DNA removal and kits for rRNA depletion. To our knowledge, this is the first report on the method of isolation of high-quality E.amylovora mRNA for RNA-seq.

Comparative transcriptome analysis of a lowly virulent strain of Erwinia amylovora in shoots of two apple cultivars – susceptible and resistant to fire blight

“…After 24 h and 6 days from the inoculation time (Fig. 6 ), samples were processed, and total RNA was isolated with a Total RNA Purification Kit (Norgen Biotek), as described by Kałużna et al [ 74 ]. At each time point, RNA was isolated separately from at least six shoots of each apple cultivar.…”

Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots

“…The plant shoots were inoculated according to the method described by Kałużna et al . 22 . Briefly, trichomes were removed from the surface of the shoots, and the shoots were punctured with a needle on approximately 7 cm of their length from the tip and covered with several 10 µl droplets of bacterial water suspension (~10 9 cfu/ml).…”

“…Total RNA was isolated (from bacterial pellet) from at least 3 shoots of each apple cultivar at each time point, immediately after cutting the shoots from the plant according to the procedure described by Kałużna and coworkers 22 . Additionally, RNA was isolated from a pure culture of E. amylovora 650 grown overnight in TY (Bacto Tryptone 0.5%, Yeast Extract 0.3%, CaCl 2 0.065%) medium, the same bacteria and growth method used for inoculation purposes.…”

“…Additionally, RNA was isolated from a pure culture of E. amylovora 650 grown overnight in TY (Bacto Tryptone 0.5%, Yeast Extract 0.3%, CaCl 2 0.065%) medium, the same bacteria and growth method used for inoculation purposes. The integrity measurements were performed according to the procedure described by Kałużna and coworkers 22 .…”

“…However, high-fidelity reactions and the accuracy of the results of gene expression profile analyses are affected by several factors, mainly associated with the standardization of pre-analytical steps 20 . On the one hand, the preparation and precise execution of the reaction itself includes (i) the careful examination of RNA extraction methods prior to their use to obtain high values of RNA integrity 21 , 22 , which may affect the results of downstream applications including RT-qPCR, and (ii) the high efficiency obtained in the reverse transcription reaction 23 , 24 . On the other hand, RT-qPCR data and obtaining credible results 11 , 25 , 26 depends on the normalization process, and the proper selection of reference genes.…”