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Nucleozol rna isolation reagent

Manufactured by Macherey-Nagel
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Sourced in Germany

NucleoZOL RNA isolation reagent is a product designed for the extraction and purification of RNA from various biological samples. It is a reagent that utilizes a guanidinium thiocyanate-phenol-chloroform extraction method to isolate total RNA.

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3 protocols using nucleozol rna isolation reagent

1

RNA Isolation, cDNA Synthesis, and qRT-PCR Analysis

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Total RNA was isolated from cultured cells or endogenous sympathetic ganglia using the NucleoZOL RNA isolation reagent (MACHEREY-NAGEL, #740404.200) according to the manufacturer’s instruction. cDNA was synthesized using the HiScript II QRT SuperMix for qPCR (Vazyme Biotech). qRT-PCR was performed using the Kapa SYBR fast qPCR master mix (Kapa) and qTOWER3G Real-Time PCR system (Analytikjena). The data were analyzed using the 2-ΔΔct calculation method. The qRT-PCR primers used are shown in Table S3.
EdU labeling was performed as previously described.32 (link) MEFs were labeled by 10 μM EdU for 12 days starting from day 1 following infection with APH lentiviruses, or for 48 hrs starting from day 14. EdU staining was carried out according to the manufacturer’s instruction (Life Technologies).
Liu S., Xiang K., Yuan F, & Xiang M. (2023). Generation of self-organized autonomic ganglion organoids from fibroblasts. iScience, 26(3), 106241.
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2

Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was isolated using the NucleoZOL RNA Isolation Reagent (Macherey–Nagel, Düren, Germany) and quantified with BioSpec-nano spectrophotometer (Shimadzu Inc.). Subsequently, cDNA synthesis and qRT-PCR were performed using the FastGene Scriptase II cDNA Synthesis 5 × Ready-Mix (NIPPON Genetics EUROPE, GmbH, Düren, Germany) and the KAPA SYBR FAST qPCR Master Mix (2X), respectively. Primers were designed using the primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Further details for the q-RT-PCR analysis are provided in [21 (link)].
Pateras I.S., Williams C., Gianniou D.D., Margetis A.T., Avgeris M., Rousakis P., Legaki A.I., Mirtschink P., Zhang W., Panoutsopoulou K., Delis A.D., Pagakis S.N., Tang W., Ambs S., Warpman Berglund U., Helleday T., Varvarigou A., Chatzigeorgiou A., Nordström A., Tsitsilonis O.E., Trougakos I.P., Gilthorpe J.D, & Frisan T. (2023). Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming. Journal of Translational Medicine, 21, 169.
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3

Quantitative Real-Time PCR Protocol

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Total RNA was extracted by NucleoZOL® RNA Isolation Reagent (MACHEREY–NAGEL, Germany). After reverse transcription by using the PrimeScript™ RT Reagent Kit (Perfect Real Time), polymerase chain reaction was conducted by an Applied Biosystems QUANT5 Studio PCR system. GAPDH was used as an endogenous control. The amplification conditions were as follows: a. denaturation at 95 °C for 10 s; b. annealing at 72 °C for 20 s; and c. extension at 60 °C for 20 s for 40 cycles. The Ct values were collated according to the results, and the 2−ΔΔCt method was used to calculate the relative expression levels of target genes. The primers used in this study are listed in Supplementary file 3.
Xie B., Lin J., Chen X., Zhou X., Zhang Y., Fan M., Xiang J., He N., Hu Z, & Wang F. (2023). CircXRN2 suppresses tumor progression driven by histone lactylation through activating the Hippo pathway in human bladder cancer. Molecular Cancer, 22, 151.
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