Masterpure yeast rna purification kit

Total RNA was prepared using the MasterPure Yeast RNA Purification Kit (Epicentre). First-strand cDNA was produced with M-MLV Reverse Transcriptase (Promega) using site-specific primers following manufacturer protocols. Real-time PCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) with SYBR Select Master Mix (Applied Biosystems). First-strand cDNA synthesis without reverse transcriptase was performed for negative controls. At least two biological repeats were performed for all experiments. Statistical analysis was performed using a student’s t test (two-tailed distribution). Error bars represent standard error of mean (s.e.m). Primers are listed in the S4 Table.

RNA was collected from 2x10⁶ cells grown for four hours in liquid YPD medium in the presence or absence of 50 μg/mL of doxycycline. Cells were removed from the medium and RNA isolated using the MasterPure Yeast RNA Purification Kit (EpiCentre, Madison, WI) according to the manufacturer’s instructions. Subsequently, 1 μg of RNA was used to synthesize cDNA using oligo-(dT)₁₈ and Superscript III reverse transcriptase (Thermo Scientific, Waltham, MA). cDNA was assayed for genomic DNA contamination using intron-spanning primers, ALO30 and ALO31, for ribosomal protein large subunit 6 (RPL6) and only cDNA lacking genomic contamination was used for qRT-PCR (S4 Table).
qRT-PCR was performed with PowerUp SYBR Green (Applied Biosystems, Foster City, CA) using an Applied Biosystems QuantStudio 3 qPCR machine and analyzed with the QuantStudio Design and Analysis Software package version 1.4.2. Primers used are listed in S4 Table. Quantification of individual TLO genes was assessed relative to ACT1. The comparative Rq method was used measure expression levels. Experiments for each gene were performed a minimum of three biological replicates in technical duplicates.

Total cellular RNA was isolated from log-phase cells using MasterPure yeast RNA purification kit (Epicentre) according to the manufacturer’s protocol. Quantification with real time RT-PCR was performed with Power SYBR Green RNA-to-CT one-step Kit (Applied Biosystems). RNA serial dilutions were used as templates to generate a standard curve of amplification for each pair of primers, and the relative concentration of the target sequence was calculated accordingly. An act1 fragment served as a reference to normalize the concentration of samples. The concentration of each target gene in wild type was arbitrarily set to 1 and served as reference for other samples.

Equivalent total cellular RNA and VLP RNA, as estimated by OD₆₀₀ or total Gag protein respectively, was extracted using the MasterPure yeast RNA purification kit (Epicentre Biotechnologies, Madison, WI) and analyzed via Northern blotting as previously described [26 (link)].

The RT-qPCR protocol was adapted from Tsuboi et al., 2020 (link). RNA was extracted using the MasterPure Yeast RNA Purification Kit (Epicentre). cDNA was prepared using ProtoScript II Reverse Transcriptase (NEB #M0368X) with a 1:1 combination of oligodT 18 primers and random hexamers (NEB) according to the manufacturer’s instructions. mRNA abundance was determined by qPCR using a home-brew recipe with SYBR Green at a final concentration of 0.5× (Thermo Fisher #S7564). Primers specific for each transcript are described in Key resources table. The mRNA levels were normalized to ACT1 abundance, and the fold change between samples was calculated by a standard ∆∆Ct analysis. All data were included except for one sample that had high technical variation, and another that had very high ACT1 CT values. Both samples were flagged as analysis began.