Expression of SEC6 in the THE1-CIp10 control strain and tSEC6 after 2 and 4 h of growth in medium (with or without DOX) was assayed using reverse transcriptase PCR (RT-PCR) (see Fig. S1 in the supplemental material). Cells from an overnight culture were resuspended in fresh YPD with or without DOX and grown for 2 or 4 h. RNA was isolated using the RiboPure yeast RNA isolation kit (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. RT-PCR was performed using the Access RT-PCR system (Promega, Madison, WI) according to the manufacturer's protocol, using primers RT-SEC6-5Det and RT-SEC6-3Det (Table 2 ) and 1 μg total RNA as the template. The absence of contaminating DNA was tested in parallel PCR-based analyses.
Ribopure yeast rna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RiboPure™ Yeast RNA Isolation Kit is a laboratory tool designed to isolate high-quality RNA from yeast samples. It utilizes a proprietary method to efficiently extract and purify RNA, making it suitable for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Access rt pcr system, by Promega (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Iscript kit, by Bio-Rad (1 mentions)
Qrt pcr mix, by Bio-Rad (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using ribopure yeast rna isolation kit
1
Analyzing SEC6 Gene Expression in Yeast
2
Yeast Salt Tolerance Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The corresponding gene sequences of the proteins were obtained by searching the MaizeSequence database (http://ensembl.gramene.org/Zea_mays/Info/Index ). The ORFs were amplified by PCR from a maize cDNA library and cloned into the yeast expression vector pYES2, which contained the Ura3 selection marker. The exogenous gene was driven by the GAL1 promoter. The construct was introduced into yeast strain YPH500 (ura3-52 lys2-801amberade2-101ochretrp1-Δ63 his3-Δ200 leu2-Δ1) according to the pYES2 vector kit instructions (Invitrogen, Carlsbad, CA, USA). Yeast salt tolerance assays were performed according to Gao et al. [16 (link)]; the NaCl concentrations were 0 M, 3 M, and 5 M. Yeast cells were collected before spotted on the agar plates, and yeast total RNAs were extracted using the RiboPure-Yeast RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA).
3
RNA Sequencing of C. parapsilosis Arachidonic Acid Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA sequencing, the C. parapsilosis GA1 strain was grown overnight in 2 ml of YPD media at 30°C with continuous shaking applied at 180 rpm. The next day, cells were washed 3 times with PBS and then counted using a Burker’s chamber. Cell concentration was adjusted to 2 × 107 cells per 10 ml PBS supplemented with 500 μM of arachidonic acid in triplicates. As a control, cells were also grown in 10 ml PBS supplemented with ethanol only, as it is the dissolving agent used for arachidonic acid solubilization. After 3 hours of growth at 30°C, RNA was isolated using the Ribopure Yeast RNA isolation Kit (Ambion) following the manufacturer’s instructions. For validating the RNA sequencing data, C. parapsilosis CLIB and GA1 cells were grown as described above and RNA extraction was performed using the same kit.
4
Molecular Cloning and Gene Expression in Fungi
5
Quantitative Analysis of CRISPR Interference in Yeast
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For qRT-PCR analysis of CRISPRi repression of PIC2 and YPR011C, 500 μL of culture was harvested after 48 hr of growth in the culturing conditions described above. The samples were centrifuged and the supernatant was discarded. RNA was extracted from the cell pellets using RiboPure™ Yeast RNA Isolation Kit (Ambion) following the manufacturer's instructions. Residual genomic DNA in the extracts was digested by DNase I (2 U/μL) (Invitrogen) at 37°C for 8 hr. SuperScript III First-Strand Synthesis System (Invitrogen) was used for cDNA synthesis with oligo-dT primers following directions from the user manual. The synthesized cDNA was diluted 1:50 and then used for SYBR qRT-PCR. Quantitative real-time PCR primers were designed using PrimerQuestTM from IDT to give products of 100 base pairs. Primer sequences are listed in Supplementary Table S. Quantitative real-time PCR was performed using iQ™ SYBR® Green Supermix and the CFX96 Touch Real-Time PCR Detection System.
6
Quantitative Gene Expression Analysis in Candida
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression analyses were performed by qRT-PCR. C. albicans strains were grown overnight in liquid YPD at 30 °C. Strains were then cultured in YPD at 30 °C (200 rpm) for 4 h. Cells were pelleted and washed three times with sterile PBS. Cell pellets were stored at −80 °C until processed for RNA isolation. RNA was isolated from Candida cells using the RiboPure Yeast RNA isolation kit (Ambion). Total RNA obtained after the elution step was subjected to DNase treatment using Turbo DNaseI. RNA concentration and quality was determined using a Nanodrop. One microgram total RNA was converted to cDNA using the iScript kit (Biorad). cDNA was diluted twofold and 1 μL was used for each PCR with the Biorad qRT PCR mix. Primers used for the expression analysis of NRG1, CUP9, MCM1 and PMA1 are listed in SI Appendix, Table S2 . PMA1 primers were used for normalization and ddCT was determined using the parental SC5314 strain (CA10) as control. Experiments were performed with three biological replicates.
7
RNA Extraction and cDNA Synthesis for C. parapsilosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA extraction, all C. parapsilosis strains were grown overnight in 2 ml of YPD medium at 30°C with shaking applied at 180 rpm. The next day, the cells were freshly inoculated in 5 ml of YPD or YPD supplemented with 100 μM/ml BPS (OD of 0.2). For gene expression analysis, strains were grown overnight in 2 ml YPD medium at 30°C with shaking applied at 180 rpm, followed by washing steps performed with 1× PBS, and were reinoculated in liquid Spider medium for incubation at 37°C for 24 h. RNA was isolated using a RiboPure yeast RNA isolation kit (Ambion) following the manufacturer’s instruction. A total of 500 ng RNA was used for cDNA synthesis. cDNA was synthesized using a RevertAid First Strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s instruction.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!