Total rna zol out d kit

Manufactured by A&A Biotechnology

Sourced in Poland

The Total RNA Zol-Out™ D kit is a reagent designed for the isolation and purification of total RNA from biological samples. It utilizes a guanidinium-based extraction method to efficiently separate RNA from other cellular components.

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6 protocols using total rna zol out d kit

Quantifying Transcript Levels by RT-qPCR

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Total RNA was isolated using TRI-REAGENT (TRI118, MRC) followed by a column-based Total RNA Zol-Out™ D kit (043, A&A Biotechnology) following the manufacturer’s protocol.
500 ng of total RNA was subjected to reverse transcription according to the protocol of the RevertAid First-Strand Synthesis Kit (K1622, TFS). Transcript quantification was performed by qPCR with Maxima SYBR Green/ROX qPCR Master Mix (K0223, TFS) on the CFX Connect Thermal Cycler System (Bio-Rad). Target gene levels were normalized to the housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were transformed as described previously [21 (link)]. Forward and reverse primers used in experiments are depicted in Table S1. If not indicated differently, results are presented as mean ± SEM for two independent biological repeats. Graphs are prepared using GraphPad Prism 8.3.0.

Sekrecka A., Kluzek K., Sekrecki M., Boroujeni M.E., Hassani S., Yamauchi S., Sada K., Wesoly J, & Bluyssen H.A. (2023). Time-dependent recruitment of GAF, ISGF3 and IRF1 complexes shapes IFNα and IFNγ-activated transcriptional responses and explains mechanistic and functional overlap. Cellular and Molecular Life Sciences, 80(7), 187.

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RNA Isolation and Expression Analysis

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To isolate RNA from HBECs and mouse lung lobes, a Total RNA Zol‐Out kit (A&A Biotechnology, Poola) and a Total RNA Zol‐Out D kit (A&A Biotechnology, Poola) were used according to the manufacturer's guidelines, respectively. To analyze relative miRNA and mRNA expression in cells and mouse lungs, RT‐qPCR and ΔΔCt calculations were used. As housekeeping genes for normalization, let7a was used for miRNA analysis, and EEF1A1 or Hprt was used for mRNA analysis. The data were compared relative to the mean value of the control group or condition, which was normalized to 1 and is indicated in each figure legend. More detailed descriptions of RNA isolation, cDNA synthesis, RT‐qPCR, and primer sequences are included in this article's Supplementary Information.

Laanesoo A., Urgard E., Periyasamy K., Laan M., Bochkov Y.A., Aab A., Magilnick N., Pooga M., Gern J.E., Johnston S.L., Coquet J.M., Boldin M.P., Wengel J., Altraja A., Bochenek G., Jakiela B, & Rebane A. (2021). Dual role of the miR‐146 family in rhinovirus‐induced airway inflammation and allergic asthma exacerbation. Clinical and Translational Medicine, 11(6), e427.

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Quantification of FMR1 mRNA and pre-mRNA Levels

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For quantification of the effect of RNase H1 insufficiency and ASOs on FMR1 mRNA and pre-mRNA level, fibroblasts were seeded on a 12-well plate and transfected at ~80% of confluency with siRNAs (future synthesis) at a final concentration of 15 nM. siRNA sequences are listed in Supplementary Table S2. After 24 h, the 11-nucleotide long (CGG^norm/- (2), CGG^norm/CGG^exp, CGG^exp/CGG^exp) or 9-nucleotide long (CGG^norm/- (1)) ASOs at 200 nM final concentration were delivered. Fibroblasts were harvested 48 h post ASOs delivery. The isolation of RNA was performed with the Total RNA Zol-Out™ D kit (A&A Biotechnology). In total, 300–500 ng of the total RNA was used for reverse transcription with GoScript^TMReverse Transcriptase (Promega) and random primers (Promega). qPCR was performed with the use of iTaq™ Universal SYBR^® Green Supermix (Bio-Rad) according to the manufacturer’s instructions and analyzed with the use of QuantStudio 7 Flex Real-Time PCR System machine (Thermo Fisher Scientific) with primers listed in Supplementary Table S2. Ct values were normalized against GAPDH. Fold differences in expression level were calculated according to the 2^−ΔΔCt method.

Derbis M., Kul E., Niewiadomska D., Sekrecki M., Piasecka A., Taylor K., Hukema R.K., Stork O, & Sobczak K. (2021). Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats. Nature Communications, 12, 1265.

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Total RNA Isolation and Reverse Transcription

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For RNA isolation, the Total RNA Zol-Out D kit (A&A Biotechnology) was used, according to the manufacturer’s protocol. In case of cells, cell pellets were suspended in 800 μL of TRI Reagent (Thermo Fisher), while mouse tissues were homogenized in 300 μL of TRI Reagent. The concentration of isolated total RNA was calculated by measurement at 260 nm using DeNovix spectrophotometer. One microgram of RNA was then used for reverse transcription using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) with random hexamer primers.

Joachimiak P., Ciesiołka A., Kozłowska E., Świtoński P.M., Figura G., Ciołak A., Adamek G., Surdyka M., Kalinowska-Pośka Ż., Figiel M., Caron N.S., Hayden M.R, & Fiszer A. (2023). Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models. BMC Biology, 21, 17.

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Total RNA Extraction from Mouse Tissues

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Mouse tissues were homogenized in TRIzol (Ambion) with 1.5 mm zirconium beads in a Bead Ruptor 12 (OMNI International). Total RNA from mouse tissues and HeLa cells were isolated by using TRIzol Reagent (Invitrogen)/TRI Reagent (Sigma-Aldrich) and the Direct-zol RNA MiniPrep Kit (Zymo Research)/Total RNA Zol-Out D Kit (A&A Biotechnology) with on-column DNase digestion according to the manufacturer’s protocol.

Sznajder L., Khan M., Tadross M., Ciesiołka A., Nutter C., Taylor K., Pearson C., Sobczak K., Lewis M., Swanson M, & Yuen R. (2023). Autistic traits in myotonic dystrophy type 1 due to MBNL inhibition and RNA mis-splicing. Research Square.

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RNA Extraction and qPCR Analysis

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RNA was purified using Qiazol (Qiagen, Germany) and Total RNA Zol-out kit (A&A Biotechnology, Poland) in case of cell cultures and Total RNA Zol-out D kit (A&A Biotechnology, Poland) in case of mouse tissue. To maintain small RNA fraction, a 1:1 ratio of isopropanol was added to the samples before loading them onto the kit columns. The concentration and quality of RNA was assessed with NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA from 300-500 ng of total RNA was synthesized using oligo(dT), RiboLock RNase
Inhibitor, RevertAid and dNTP Mix according to the manufacturer's protocol (Thermo Scientific, USA). Real-time qPCR (RT-qPCR) was performed in triplicates with 5xHOT FIREPol EvaGreen qPCR Supermix (Solis BioDyne, Estonia) and the primers from TAG Copenhagen (Supplementary Table S1) using ViiA™ 7 Real-Time PCR system (Applied Biosystems, USA). The relative gene expression levels were normalized according to the level of elongation factor 1 alpha (EF1A) in case of cell cultuers and beta-2 microglobulin (B2M) in case of mouse samples J o u r n a l P r e -p r o o f and calculated using the comparative Ct (ΔΔCt) method. The mean level of non-transfected cells or control experiments was equalized to 1.

Carreras-Badosa G., Maslovskaja J., Periyasamy K., Urgard E., Padari K., Vaher H., Tserel L., Gestin M., Kisand K., Arukuusk P., Lou C., Langel Ü., Wengel J., Pooga M, & Rebane A. (2020). NickFect type of cell-penetrating peptides present enhanced efficiency for microRNA-146a delivery into dendritic cells and during skin inflammation. Biomaterials, 262.

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