Computational protocol: Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

Similar protocols

Protocol publication

[…] RNA extractions for RT-PCRs conducted to support EST data were carried out on pelleted haploid cells grown in CM or MN, or filamentous diploids or dikaryons harvested from plates. The tissue from haploid or mycelial cell types was frozen in liquid nitrogen and homogenized using a mortar and pestle. Vacuum-dried teliospores were disrupted following the protocol described in Zahiri et al. (). For all other experiments, U. maydis haploid cells, U. maydis germinating teliospores, or U. hordei haploid cells, were pelleted by centrifugation. These cells, or U. hordei teliospores (collected from field samples), were resuspended in TRIzol reagent (Invitrogen), and transferred into 2 ml screw-cap tubes containing Lysing Matrix C (MP Biomedicals). Cells were disrupted as described in Zahiri et al. (). Alternatively, U. hordei-infected barley heads were ground in liquid nitrogen immediately prior to RNA extraction and resuspended in TRIzol reagent (Invitrogen). Following cell disruption, RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA samples were precipitated, treated with DNase I, screened for genomic DNA contamination and assessed for quality as described in Morrison et al. ().For first-strand synthesis reactions, 200 ng of DNase I-treated total RNA was used as template. These reactions were primed with oligo-d(T)16, a sense-specific primer, an antisense-specific primer, a tagged strand-specific primer or water (to account for false-priming). Primers were designed for sense- or antisense-specific first-strand synthesis reactions using Primer3 (Rozen and Skaletsky, ) following the protocol outlined in Ho et al. (). The strand-specific primer sequences are listed in . All first-strand synthesis reactions were carried out using the TaqMan Gold RT-PCR kit (Applied Biosystems) with the conditions outlined in Ho et al. (). Following first-strand synthesis, cDNA was diluted fourfold (1:3) with DEPC-treated water.To detect dsRNA, 2.5 μg of DNase I-treated total RNA was incubated in S1 nuclease digestion reactions at 37°C for 30 min. In separate reactions, the final concentration of S1 Nuclease (Invitrogen) included: 0, 0.01, 0.1 or 1 U μl−1. Next, dsRNA was phenol/chloroform extracted, precipitated with NH4Ac/Ethanol/GlycoBlue Coprecipitant (Invitrogen) at −20°C for 60 min, and resuspended in 15 μl DEPC-treated H2O. Two microlitres of S1 trimmed RNA was used as template in first-strand synthesis reactions. [...] All primers used in RT-PCRs to analyse transcript expression were designed using Primer3 (Rozen and Skaletsky, ) and are listed in . Two microlitres of diluted cDNA was used as template for each RT-PCR. All RT-PCRs were performed using Amplitaq Gold DNA polymerase (Applied Biosystems) and 30 or 35 cycles. RT-PCRs with 30 cycles were considered to be semi-quantitative because equal amounts of total RNA were used as template for each first-strand synthesis reaction, and equal volumes of cDNA were used as template for each RT-PCR. One quarter of the final product mixture was electrophoretically separated on an agarose gel (1× TAE), and visualized by ethidium bromide staining.TaqMan minor groove binder (MGB) probes were designed using Primer Express v2.0 (Applied Biosystems) and are listed in . Two microlitres of diluted cDNA was used as template for each quantitative PCR (RT-qPCR). All RT-qPCRs were performed using the TaqMan Universal PCR Master Mix (Applied Biosystems). Data were collected and analysed on an ABI PRISM 7900HT using Sequence Detection System Version 2.1 (Applied Biosystems). Relative transcript levels for um02151 were measured for eight independent FB1 [pCMas-um02151] strains, compared with an average of four independent FB1 [pCM768] strains using the ΔΔCT method. Three technical replicates for each sample were performed and umgapd was used as an internal standard. […]

Pipeline specifications

Software tools Primer3, Primer Express
Application qPCR
Organisms Zea mays, Ustilago maydis
Diseases Infection