Total ribonucleic acid (RNA) was extracted from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Copy DNA was synthesized from 1 µg RNA using FastGene Scriptase II kit (NIPPON Genetics Europe, Dueren, Germany) and random hexamer primers. The primer sequences are described in Supplemental Table S1 (38 (link)). The polymerase chain reactions were performed using Fast SYBR Green Master Mix or TaqMan Fast Advanced Master Mix (Applied Biosystems, Foster City, CA, USA). For quantification of gene expression, Applied Biosystems StepOnePlus Real-Time PCR system was used. The relative expression levels of the target genes were calculated as a ratio to the hypoxanthine guanine phosphoribosyl transferase (Hprt) gene.
Fastgene scriptase 2 kit
Manufactured by Nippon Genetics
Sourced in Germany
The FastGene Scriptase II kit is a reverse transcription kit designed for the synthesis of complementary DNA (cDNA) from RNA templates. The kit contains all the necessary components, including an RNA-dependent DNA polymerase (reverse transcriptase), buffer, and reagents, to efficiently convert RNA into first-strand cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Fast sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Quantinova sybr green kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
6 protocols using fastgene scriptase 2 kit
1
Quantitative RT-PCR for Gene Expression
2
RNA Binding Analysis by RIP Assay
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RNA co-IP (RIP) assays were performed to analyze the RNA binding of target proteins as described earlier (3 (link)). Cells were lysed in RIP buffer [25 mM Tris–HCl pH 7.5, 2 mM MgCl2, 150 mM NaCl, 0.5 mM DTT, 0.2% Triton X-100, protease inhibitor, 0.2 mM phenylmethylsulfonyl fllu- oride (PMSF) and 0.02 U/ml Ribolock] with glass beads utilizing a FastPrep cell homogenizer. GFP-trap beads were used to pull-down GFP- or MYC-tagged proteins. Samples were split after washing. One part was loaded on an SDS gel and checked for pulldown on a western blot, while the other part was used for RNA isolation and RNA binding analyses by qPCR. RNAs from both the lysate and the eluate were extracted with Trizol and purified with phenol–chloroform. cDNAs were synthesized with the FastGene Scriptase II Kit (NIPPON Genetics) by using random hexamer primers.
3
Cytosolic Fractionation Assay for mRNA Leakage
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The cell fractionation assay was carried out to examine leakage of 3′-extended faulty mRNAs in the cytosol as described earlier (39 (link)). Following harvest, the logarithmic cells were washed with H2O and YPD/1 M sorbitol/2 mM DTT, respectively, and then digested with zymolyase in YPD/1 M sorbitol/1 mM DTT for 10–30 min to obtain spheroplasts. After 30 min of recovery in YPD/1 M sorbitol at 25°C, cells were shifted to 37°C for 3 h to induce the mutant phenotype. Then, cells were lysed and the cytosolic fraction was obtained in the supernatant of the Ficoll density gradient after centrifugation. Antibodies of the nuclear proteins Nop1 or Yra1 and the glycolytic enzyme Zwf1 were used to confirm the successful separation of the cytosolic fraction on the western blot. Total RNA was isolated from both the lysate and the cytosolic fraction with the NucleoSpin RNA kit (MACHEREY-NAGEL). cDNAs were synthesized with the FastGene Scriptase II Kit (NIPPON Genetics) by using random hexamer primers. Leakage of readthrough mRNAs was analyzed via qPCR with primers flanking the cleavage site.
4
Isolation and qRT-PCR Analysis of pDCs
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pDCs were isolated from spleen using MACS and PDCA-1-beads (Miltenyi) and RNA was isolated by using RNeasy Micro Plus Kit (QIAGEN). cDNA was synthesized using a reverse transcriptase system (FastGene Scriptase II kit; Nippon Genetics). qRT-PCR was performed with a SYBR Green based kit (QuantiNova SYBRGreen Kit; QIAGEN) on a AriaMx Realtime machine (Agilent). Actin was used as the housekeeping gene. The following primers were used:
5
DNA/RNA Isolation from Chroomonas gyreus
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For DNA/RNA isolation, G. theta was cultured in 200 ml f/2 medium for 7–14 days, as described earlier (Mix et al., 2018 (link)). G. theta cells were centrifuged at 3,000 x g and 21°C for 10 min. Total DNAs were isolated from the pelletized G. theta cells via the CTAB method and were stored at −20°C. Total RNAs of G. theta were extracted using the RNeasy Mini kit (Qiagen) and were promptly treated with DNAseI (Thermo Fisher Scientific), following the manufacturer's instructions, to remove potential genomic DNA contamination. cDNA was synthesized with the FastGene Scriptase II Kit (Nippon Genetics) using random hexamer oligonucleotides provided by the manufacturer. Synthesized cDNA was used for amplification of gene sequences with specific oligonucleotides (refer to Supplementary Table S4 ).
6
Quantitative Gene Expression in Mouse Tissues
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Aorta, right femur and levator ani muscle collected at euthanasia were snap-frozen in liquid nitrogen and stored at -80°C until further processing. The bone marrow fraction of the femur was removed by centrifugation. Total RNA was extracted from tissues using RNeasy kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. cDNA was synthesized from 1 µg RNA using the FastGene Scriptase II kit (NIPPON Genetics Europe, Dueren, Germany) and random hexamer primers. The PCR reactions were performed using Fast SYBR Green Master Mix and the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression was normalized for Actb and Gapdh housekeeping genes and expressed relative to the control group (2 -∆∆Ct method). The following primer sequences were used: Actb (5'-CATTGCTGACAGGATGCAGAAGG-3' ; 5'-TGCTGGAAGGTGGACA GTGAGG-3'), Gapdh (5'-TCTTGTGCAGTGCCAGCCTC-3' ; 5'-TGAAGGGGTCGTTGATGGCAA-3'), Ar (5'-AA GAGCTGCGGAAGGGAAAC-3' ; 5'-ACATTTCCGGAGACGACACGA-3'); Acta1 (5'-GAACCCCAAAGCTAACC GGG-3' ; 5'-ATCCAACACGATGCCGGTG-3'); Col1a1 (5'-GCATGGCCAAGAAGACATCCC-3' ; 5'-CATAGCAC GCCATCGCACAC-3') and Fkbp5 (5'-TAACTTGGGCGACCCTCACC-3'; 5'-ACTTCTGGCTCGGAACCCTG-3').
All primers were designed to hybridize to different exons, and generation of single correct amplicons was checked by melting curve dissociation.
All primers were designed to hybridize to different exons, and generation of single correct amplicons was checked by melting curve dissociation.
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