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TranScriba Kit

Manufactured by A&A Biotechnology
Sourced in Poland

The TranScriba Kit is a laboratory equipment product designed for RNA extraction and purification. It provides a standardized protocol and necessary reagents to isolate high-quality RNA from various biological samples.

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35 protocols using TranScriba Kit

Total RNA was isolated from 0.5 g of lyophilized cyanobacteria samples using the RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s instructions. The RNA was then transcribed into cDNA; cDNA was obtained from 5 µg RNA using the TranScriba Kit (A&A Biotechnology), according to the manufacturer’s instructions. Universal primers for amplification of 16S rRNA in cyanobacteria were used to find the 16S rRNA fragments [58 (link)]; forward primer CYA 359F: 5′-GGGGAATYTTCCGCAATGGG-3′and reverse primer CYA 781R: 5′-GACTACWGGGGTATCTAATCCCWTT-3′. The PCR products obtained were subjected to sequencing (Genomed S.A., Warszawa, Poland). The 16S rRNA sequences (fragments) are shown in Supplementary Material Notes S1. Subsequently, the sequences were used for the reconstruction of the 16S rRNA tree.
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The mRNA level of the ABCB1 gene was assessed using quantitative real-time PCR. Briefly, HL-60 cells were seeded in 6-well plates (4.0 × 105 cells/mL) in 2 mL of growth medium and treated with 5a for 24 h. Total RNA was extracted using the Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. The concentration of RNA used for further experiments was 500 ng. cDNA was synthesized using Transcriba Kit (A&A Biotechnology, Gdynia, Poland). The amplification of cDNA was performed using RT PCR Mix SYBR (A&A Biotechnology, Gdynia, Poland) and gene specific primers (Table 3) in the Stratagene Mx3005P QPCR System (Agilent Technologies, Inc. Santa Clara, CA, USA) according to the manufacturer’s guidelines. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The expression level of ABCB1 gene was determined by the 2−∆∆CT method [23 (link)].
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On day 2 of differentiation, total RNA from 3T3-L1 cells was extracted with Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. First-strand cDNA synthesis was performed with 1 μg of total RNA using a TranScriba Kit (A&A Biotechnology, Poland). Quantification of gene expression in the 3T3-L1 cells treated with COST was measured using a real-time PCR system (SmartCycler DX real-time PCR System Cepheid, USA). PCR was performed in a final volume of 20 μl, including 10 ng of sample cDNA, 5 μM of specific forward and reverse primers, and 15 μl of Real Time 2xPCR Master Mix EvaGreen. The reaction mixtures were incubated for an initial denaturation at 93 °C for 3 min, followed by 45 PCR cycles of 1 min at 93 °C, 1 min at 60 °C and 1 min at 72 °C.
PPARγ:
(+)5′-GCCCTTTGGTGACTTTATGG-3′, (−) 5′-CAGCAGGTTGTCTTGGATGT-3′;
C/EBP:
(+)5′-TTGCACCTCCACCTACATCC-3′, (−) 5′-CCACAAAGCCCAGAAACCTA-3′. The relative amount of each gene was calculated using the 2−ΔΔCt method.
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Strains obtained by transformation of PAO1161 with pMEB12 or pKGB8 were used for total RNA preparation. RNA isolation and sequencing as well as data analysis were performed essentially as previously described [79 (link)]. Raw data are available in the NCBI’s Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/ (accessed on 30 October 2021)) under accession number GSE186749.
Expression changes for selected genes were confirmed by RT-qPCR using RNA isolated from independent cultures. In total, 1 µg of each RNA sample was used for reverse transcription (TranScriba Kit, A&A Biotechnology, Gdańsk, Poland) with the use of random hexamers. Three technical replicates of PA2577 overproducer and EV control were used. The nadB was used as the reference. RT-qPCR was also used for examination of the expression of PA2577 and PA2576 in the early (OD600~0.5) and late exponential (OD600~1.5) phases of growth of P. aeruginosa WT cultures and for PA2576 also in P. aeruginosa ΔPA2577 strain. The rpsL gene was used as the reference. Relative gene expression was calculated using the Pfaffl method [80 (link)]. All oligonucleotides used in RT-qPCR are listed in Table A2.
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Briefly, the cells were plated into 6-well culture dishes (3 × 105 cells/well) for 24 h prior to the addition of MtRV plant extract (1.5 mg mL−1). The total RNA isolation kit (A&A Biotechnology, Gdynia, Poland) was used to isolate total RNA from cells treated with the plant extracts. The obtained RNA was transcribed into cDNA using TranScriba Kit (A&A Biotechnology). Following this, the expression of four genes (Bax, Bcl-2, Cas-3, TP53) was measured by qRT-PCR using TaqMan® Real-Time PCR Master Mix (Life Technologies, Carlsbad, CA, USA) and Agilent Technologies Stratagene Mx300SP (Santa Clara, CA, USA) working on MxPro software. TaqMan probes (Life Technologies, CA, USA) were used to analyse genes and 18S RNA (Life Technologies) was included as a reference gene. The PCR was performed as follows: 95 °C for 10 min, 30 cycles of 95 °C for 15 s and 60 °C for 60 s.
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RNA isolation
and purification, reverse transcription, and qPCR were
performed according to previously published data.27 (link) Briefly, RNA was isolated and purified from the S. aureus 5 N isolate (OD600 = 0.5) after
samples were treated with sublethal doses of aPDI (reduction in bacterial
cell count ∼0.5 log10 units) using a Syngen Blood/Cell
RNA Mini Kit (Syngen, Poland). The TranScriba kit (A&A Biotechnology,
Poland) was used to transcribe the RNA to complementary DNA (cDNA).
qPCR assays were performed using a LightCycler 480 II (Roche Life
Science, Germany). The reaction mixture (10 μL total) consisted
of 5 μL of Fast SG qPCR Master Mix (EURx, Poland), 200–400
nM of each primer (TIB MOLBIOL, Germany) (Table S1), nuclease-free water, and 1 μL of fivefold dilution
of cDNA. The following steps were implemented (Table S2). Melting curve analysis was carried out to exclude
primer-dimer formation or nonspecific amplification. Relative changes
in the expression of the sec, tst, srrA, and srrB genes were normalized
to the gmk reference gene.
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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.
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Purified bovine serum albumin (BSA), T6P dipotassium salt, uridine 5′-diphosphoglucose disodium salt from Saccharomyces cerevisiae (UDPG), d-glucose-6-phosphate disodium salt hydrate (G6P), and alkaline phosphatase from bovine intestinal mucosa (lyophilized powder, 10–30 defined enzyme activity (DEA) units/mL solid), penicillin G sodium salt, nystatin, IVM, 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), agarose, and ethidium bromide were purchased from Sigma-Aldrich (Germany and USA). Total RNA Kit, TranScriba Kit, and SYBR Green B PCR-MIX Taq were obtained from A&A Biotechnology (Poland). The components of 0.1 M acetic acid-ammonia buffer and 0.1 M phosphoric buffer (resp., pH 4.2 and pH 7.0) (acetic acid, ammonia, NaH2PO4, and Na2HPO4  × 7H2O2) and of Ascaris Ringer's Solution (ARS) medium (KCl, CaCl2  × 2H2O, MgCl2  × 6H2O, NaCl, and sodium acetate) were of high purity grade and were purchased from Chempur (Poland) and P.P.H. Stanlab (Poland).
The water used for the analysis was deionized with the use of a Direct-Q Ultrapure UV3 Water System (EMD Millipore, USA). The water, media, sodium saline, and surgical instruments were sterilized using a Classic Standard Prestige Medical autoclave (Ma-Je-R, Poland).
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9

RNA Extraction and RT-PCR Analysis of Chondrogenic Markers

Three constructs from each time point were dissolved in 100 mM sodium citrate, containing 0.08 U∙μL−1 Proteinase K and 1.0 U∙μL−1 RNAse Inhibitor (A&A Biotechnology), while shaking for 5 minutes at 37°C, followed by ribonucleic acid (RNA) isolation with TriReagent (Sigma- Aldrich). Chloroform was then added, and the probes were centrifuged at 12,000 RCF for 15 minutes at 4°C. The supernatant was collected and mixed with a 1:1 volume of cold 99% ethanol. The solution was then transferred to the columns from RNeasy Mini Kit. The isolation steps were performed according to the RNeasy Mini Kit manual. The RNA concentration was measured using the Qubit 4 Fluorometer. For reverse transcription polymerase chain reaction (RT-PCR), TranScriba Kit (A&A Biotechnology) was used with random hexamer primers and 300 ng of total RNA. The following genes for real-time PCR were selected: COL1A1, COL2A1, COL10A1, SOX9, and RUNX2, with GAPDH as the housekeeping gene. The designed starters are shown in Table 2. The QuantStudio 6k Flex Real-Time PCR System (Applied Biosystems) with 1 μL of complementary deoxyribonucleic acid (cDNA) and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) was used to evaluate the expression of these genes. Primers were used at a final concentration of 0.5 μM. The gene expression results were tested with the twoway analysis of variance (ANOVA).
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To transcribe RNA to complementary DNA (cDNA), a TranScriba kit (A & A Biotechnology, Poland) was used. One hundred nanograms of RNA was reverse transcribed with 1 µL of dN-hexamer in a total reaction volume of 20 µL according to the manufacturer’s instructions. The cDNA synthesis conditions were as follows: pre-incubation for 5 min at 25 °C, elongation of hexamers for 60 min at 42 °C, termination for 5 min at 70 °C and cooling at 4 °C. The cDNA samples were stored at − 20 °C for later use.
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