Bead beat total rna mini kit

Manufactured by A&A Biotechnology

Sourced in Poland

The Bead-Beat Total RNA Mini kit is a product designed for the rapid and efficient extraction of total RNA from a variety of sample types. The core function of this kit is to provide a simple and effective method for isolating high-quality RNA for downstream applications.

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Lab products found in correlation

Nanodrop, by Thermo Fisher Scientific (4 mentions) Transcriba kit, by A&A Biotechnology (3 mentions) 7500 real time pcr thermal cycler, by Thermo Fisher Scientific (2 mentions) Nextseq 500 sequencer, by Novogene (2 mentions) Second strand synthesis buffer, by Illumina (2 mentions) Clean up rna concentrator, by A&A Biotechnology (2 mentions) 2100 bioanalyzer, by Agilent Technologies (2 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions) Primer expert software, by Thermo Fisher Scientific (1 mentions) Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions) Novaseq 6000, by Illumina (1 mentions)

7 protocols using bead beat total rna mini kit

Quantitative PCR Analysis of Y. lipolytica

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Total RNA was extracted from Y. lipolytica according to the manufacturer’s instructions using the Bead-Beat Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland). The TranScriba kit (A&A Biotechnology, Gdynia, Poland) was used to synthesize the cDNA strands.
Quantitative PCR (qPCR) was performed in a 7,500 real-time PCR thermal cycler (Applied Biosystems, Waltham, MA, USA) using an SYBR®Green B PCR MasterMix (A&A Biotechnology, Gdynia, Poland). Each reaction contained 0.5 μl of cDNA template, 5 μl of SYBR®Green B PCR MasterMix, and 0.5 μl of forwarding and reversed primers, which were made up to 10 μl using ddH₂O. Reaction conditions were as follows:95°C for 3 min, 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, 2–4 steps of 40 cycles, and melting curve phases: 94°C for 15 s, 60°C for 60 s, 95°C for 30 s, 60°C for 15 s. Each qPCR reaction was performed in technical replicates.

Yang S., Pan X., Wang Q., Lv Q., Zhang X., Zhang R, & Rao Z. (2022). Enhancing erythritol production from crude glycerol in a wild-type Yarrowia lipolytica by metabolic engineering. Frontiers in Microbiology, 13, 1054243.

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Quantitative analysis of gene expression

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RNA extraction and purification were conducted using the Bead-Beat Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland) after mechanical disruption of the cells. For reverse transcription, a TranScriba Kit (A&A Biotechnology) was used with 5 ng of cDNA as a matrix. Real time quantitative PCR reaction (RT-qPCR) targeting YFP, SoA, TlG and actin encoding genes was conducted using RT PCR Mix SYBR^® B (A&A Biotechnology) and StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) apparatus. Actin-encoding gene (ACT1) was used as an internal calibrator for the normalisation of target gene expression. Cells from 0 h of the cultures were used as external calibrators. Data were processed according to the standard ΔΔCt method. Primers specific to actin and target genes are listed in Table S2.

Gorczyca M., Kaźmierczak J., Fickers P, & Celińska E. (2022). Synthesis of Secretory Proteins in Yarrowia lipolytica: Effect of Combined Stress Factors and Metabolic Load. International Journal of Molecular Sciences, 23(7), 3602.

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Intracellular Stress-Related Gene Expression

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Total RNA isolation, reverse transcription (RT) reaction and real-time quantitative PCR (RTqPCR) were conducted as described by Borkowska et al. (2020 ). Briefly, Bead-Beat Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland) and MixerMill MM400 (Retsch GmbH, Haan, Germany) were used for isolation of RNA. TranScriba Kit (A&A Biotechnology, Gdynia, Poland) and a Veriti Thermal Cycler (Applied Biosystems, USA) were used for the first cDNA strand synthesis. The RTqPCR was carried out using SYBR®Green PCR MasterMix kit B (A&A Biotechnology, Poland), FrameStar® 96 Well Semi-Skirted PCR Plates and a 7500 Real-time PCR Thermalcycler (Applied Biosystems, Foster City, USA). Real-time PCR primers targeting the intracellular stress-related genes and the internal calibrator were designed with Primer Expert Software (Applied Biosystems, Foster City, USA) (Supplemental Table S1). Comparative gene expression analysis of the treated samples was conducted vs the control culture (Fig. 1). Data analysis was carried out according to the delta-delta Ct (ΔΔCt) method (Livak and Schmittgen 2001 (link)).

Kubiak-Szymendera M., Skupien-Rabian B., Jankowska U, & Celińska E. (2021). Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica—molecular background revealed by quantitative proteomics. Applied Microbiology and Biotechnology, 106(1), 349-367.

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Yeast RNA Sequencing Protocol

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The original liquid cultures were grown overnight in SC-ura or SC-leu, transferred to YPD, and grown to the log phase (OD600 = 0.6). Total RNA was extracted from yeast cells using the Bead-beat Total RNA Mini kit (A&A Biotechnology, Gdańsk, Poland, catalogue no. 031-25BB) and purified using Clean-Up RNA Concentrator (A&A Biotechnology, catalogue no. 039-25C). RNA quality control was checked with Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
RNA sequencing and data quality filtration were performed by the Hemispherian company with the following procedure: mRNA was enriched using oligo(dT) beads. The mRNA was fragmented randomly, and then cDNA was synthesized using an mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H, and DNA Pol I were added to initiate second-strand synthesis. After a series of terminal repair and ligation of sequencing adaptors, the double-stranded cDNA library was completed through size selection and PCR enrichment.
Whole-transcriptome RNA-seq was performed using an Illumina NextSeq-500 sequencer (Novogene). Raw data that comprised three biological replicates for each strain were processed by removing reads that contained adapters and low-quality reads. The reads, having passed both operations, comprised 96% of the initial data, with an average read length of 150 bases.

Rudzińska I., Cieśla M., Turowski T.W., Armatowska A., Leśniewska E, & Boguta M. (2021). Reprogramming mRNA Expression in Response to Defect in RNA Polymerase III Assembly in the Yeast Saccharomyces cerevisiae. International Journal of Molecular Sciences, 22(14), 7298.

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Total RNA Extraction from Biomass

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Total RNA was extracted from the biomass samples collected from the steady-state culture. The RNA was isolated using Bead-Beat Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. Extracts were obtained by mechanical disruption of the cells using Mixer Mill MM400 (Retsch, Germany) at 30 Hz for 30 s. The RNA extracts were verified for quantity using spectrophotometer (NanoDrop, Thermo Fisher Scientific) and for quality through agarose gel electrophoresis [33 ].

Korpys-Woźniak P, & Celińska E. (2021). Global transcriptome profiling reveals genes responding to overproduction of a small secretory, a high cysteine- and a high glycosylation-bearing protein in Yarrowia lipolytica. Biotechnology Reports, 31, e00646.

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Yeast RNA Extraction and NGS Sequencing

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Steady-state-maintained yeast cells were harvested and processed according to a Bead-Beat Total RNA Mini Kit protocol (A&A Biotechnology, Gdynia, Poland) to extract total RNA. The quality of RNA was verified through agarose gel electrophoresis [33] and spectrophotometry (NanoDrop, Thermo Fisher Scientific). Library construction and NGS RNA sequencing were executed using NovaSeq 6000 (Illumina) apparatus at Genomed S.A. (Warsaw, Poland). Raw sequencing reads are available on the NCBI Sequence Read Archive under the BioProject number PRJNA869113 (see Data Availability section).

Korpys-Woźniak P, & Celińska E. (2023). Molecular background of HAC1-driven improvement in the secretion of recombinant protein in Yarrowia lipolytica based on comparative transcriptomics. Biotechnology Reports, 38, e00801.

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Yeast RNA-seq Library Preparation

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The original liquid cultures were grown overnight in SC-ura or SC-leu, transferred to YPD, and grown to the log phase (OD600 = 0.6). Total RNA was extracted from yeast cells using the Bead-beat Total RNA Mini kit (A&A Biotechnology, Gda ńsk, Poland, catalogue no. 031-25BB) and purified using Clean-Up RNA Concentrator (A&A Biotechnology, catalogue no. 039-25C). RNA quality control was checked with Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
RNA sequencing and data quality filtration were performed by the Hemispherian company with the following procedure: mRNA was enriched using oligo(dT) beads. The mRNA was fragmented randomly, and then cDNA was synthesized using an mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H, and DNA Pol I were added to initiate second-strand synthesis. After a series of terminal repair and ligation of sequencing adaptors, the doublestranded cDNA library was completed through size selection and PCR enrichment.
Whole-transcriptome RNA-seq was performed using an Illumina NextSeq-500 sequencer (Novogene). Raw data that comprised three biological replicates for each strain were processed by removing reads that contained adapters and low-quality reads. The reads, having passed both operations, comprised 96% of the initial data, with an average read length of 150 bases.

Rudzińska I. (2020). Reprogramming of mRNA expression in response to a defect in RNA polymerase III assembly in yeast Saccharomyces cerevisiae.

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