Total RNA from tachyzoites was extracted using Total RNA Mini Plus (A&A Biotechnology) according to the manufacturer’s instruction. The isolated RNA was concentrated using Clean-Up RNA Concentrator (A&A Biotechnology) and stored at -80°C. Single strand cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) and stored at -20°C.
Clean up rna concentrator
Manufactured by A&A Biotechnology
Sourced in Poland
The Clean-Up RNA Concentrator is a laboratory equipment designed for the purification and concentration of RNA samples. It utilizes a filtration process to remove impurities and concentrate the RNA, without introducing bias or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Eco real time pcr system, by Illumina (3 mentions)
Fenozol reagent, by A&A Biotechnology (3 mentions)
Aceq qpcr sybr green mix, by Vazyme (3 mentions)
Total rna mini plus, by A&A Biotechnology (2 mentions)
M mlv polymerase, by Promega (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 sequencer, by Novogene (2 mentions)
Second strand synthesis buffer, by Illumina (2 mentions)
Bead beat total rna mini kit, by A&A Biotechnology (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Total rna mini plus kit, by A&A Biotechnology (1 mentions)
Transcriba kit, by A&A Biotechnology (1 mentions)
Ds 11 fx spectrophotometer fluorometer, by DeNovix (1 mentions)
M mlv transcriptase, by Promega (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
8 protocols using clean up rna concentrator
1
Isolation and Reverse Transcription of Tachyzoite RNA
2
RNA Extraction and Reverse Transcription
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Total RNA was isolated from the overnight culture (control) and strains subjected to the ceviche preparation process using the Total RNA Mini Plus Kit (A&A Biotechnology, Gdynia, Poland) and purified and concentrated according to the manufacturer’s instructions using the CleanUp RNA Concentrator (A&A Biotechnology, Gdynia, Poland). RNA integrity of all samples was checked by loading 10 µL RNA into a 1.2% agarose gel in 0.5% TBE buffer and running at 90 V for 1 h. Once the RNA integrity is confirmed by visualizing the two bands (16S and 23S RNA) with little smearing. RNA concentration and purity were measured optically using a DeNovix DS11 FX spectrophotometer/fluorometer (DeNovix Inc., Wilmington, NC, USA) based on sample absorption at wavelengths of 260 nm and 280 nm. All RNA samples were immediately partially reverse-transcribed and the rest stored at −80 °C. All the RNA samples were normalized to 5 µg/µL and transcribed into the cDNA using the TranScriba kit (A&A Biotechnology, Gdynia, Poland) for the synthesis of first-strand cDNA. This kit uses recombinant MMLV reverse transcriptase, which has low RNAseH activity at 37–42 °C and optimal DNA polymerase activity. Template RNA was protected with a recombinant RNAse inhibitor. Random sequence hexamer was used as a primer.
3
Quantification of Gene Expression by RT-qPCR
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Total RNA was isolated by the modified guanidinium isothiocyanate and phenol/chloroform extraction using Fenozol reagent (A&A Biotechnology), followed by DNAse I digestion and purification using Clean-Up RNA Concentrator (A&A Biotechnology) as recommended by the manufacturer. Equal amounts of RNA (1 μg) were reverse-transcribed using M-MLV transcriptase (Promega) and oligo(dT)15 primer according to manufacturer’s recommendations. RT-qPCR was performed using AceQ qPCR SYBR Green Mix (Vazyme Biotech) on Eco Real-Time PCR System (Illumina). Reaction for each data point was performed in duplicates. RNA expression was normalized to a geometric mean of at least two of the following reference genes: eEF2, Tbp, PolR2b, ActB. Primes used for qPCR experiments are listed in Supplementary Table 2 .
4
Liver RNA Extraction and mTOR Gene Expression
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The total tissue RNA was isolated from the liver sample with a use of standard commercial kit (Total RNA mini plus, A&A Biotechnology, Poland). DNAse I treatment with Clean-up RNA concentrator (A&A Biotechnology, Poland) was applied to remove traces of DNA from all samples. RNA purity was checked using the NanoDrop 2000 (ThermoFisher). Primer specific, high capacity cDNA reverse transcription kit was used to convert RNA to cDNA (TranScriba 1 step PCR Mix, A&A Biotechnology, Poland). All RNA samples were adjusted to a starting concentration of 50 ng/µL. The expression levels of mTOR and β-actin genes (as a reference) were estimated with SYBR Green on real-time PCR (Applied Biosystems). ΔCt values for mTOR in experimental groups were corrected for untreated controls with average ΔCt as the calibrator, according to 2-ΔΔCt method.33 (link)
5
Total RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated by the modified phenol/chloroform extraction method using a Fenozol reagent (A&A Biotechnology) and then treated with TURBO DNase (Thermo Scientific) followed by column purification with the Clean Up RNA Concentrator (A&A Biotechnology). Equal amounts of RNA (1 μg) were reverse transcribed with a mixture of oligo(dT)15 and random hexamer primers using M-MLV polymerase (Promega). qRT-PCR gene expression quantifications were performed using AceQ qPCR SYBR Green Mix (Vazyme Biotech) on an Eco Real-Time PCR System (Illumina, San Diego, CA, USA). RNA expression was normalized to a geometric mean of two reference genes: Eef2 and Polr2b for mouse cell lines and EEF2 and TBP for human cell lines. All sequences of primers are listed in Table S4 .
6
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.
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.
7
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.
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.
8
RNA Isolation and RT-qPCR Quantification
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Total RNA was isolated by the modified phenol/chloroform extraction method using Fenozol reagent (A&A Biotechnology), then treated with TURBO DNase (Thermo Scientific) followed by column purification with Clean Up RNA Concentrator (A&A Biotechnology). Equal amounts of RNA (1 µg) were reverse-transcribed with a mixture of oligo(dT)15 and random hexamer primers using M-MLV polymerase (Promega). RT-qPCR gene expression quantifications were performed using AceQ qPCR SYBR Green Mix (Vazyme Biotech) on Eco Real-Time PCR System (Illumina, San Diego, CA). RNA expression was normalized to a geometric mean of two reference genes:
Eef2 and Polr2b for mouse cell lines and EEF2 and TBP for human cell lines. All sequences of pimers are listed in Manuscript supplement-Table I.
Eef2 and Polr2b for mouse cell lines and EEF2 and TBP for human cell lines. All sequences of pimers are listed in Manuscript supplement-Table I.
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