The protocol for real-time RT-PCR has been described previously [35 (link)]. Total RNA from drug-treated or untreated cells was isolated using an RNeasy® Mini Kit (QIAGEN) according to the manufacturer’s instructions. Transcription of RNA into cDNA was performed using a PrimeScript ™ 1st Strand cDNA Kit (Takara, Japan). The following primers for FCV Pro-Pol and GAPDH were designed: FCV-for, 5’-ATGATTTGGGGTTGTGATGT-3’; FCV-rev, 5’-TGGGGCTRTCCATGTTGAT-3’; GAPDH-for, 5’-TGACCACAGTCCATGCCATC-3’; GAPDH-rev, 5’-GCCAGTGAGCTTCCCGTTCA-3’. The procedure for PCR consisted of an initial step at 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 15 s. The relative level of RNA expression was determined by the 2-∆∆CT method [41 (link)]. GAPDH mRNA was analyzed as a loading control.
Primescript 1st strand cdna kit
Manufactured by Takara Bio
Sourced in Japan
The PrimeScript™ 1st Strand cDNA Synthesis Kit is a reverse transcription kit for the synthesis of first-strand cDNA from total RNA or mRNA templates. It contains the necessary components for efficient cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Phusion high fidelity pcr master mix with gc buffer, by New England Biolabs (1 mentions)
Dynabeads mrna direct purification kit, by Thermo Fisher Scientific (1 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Mircury lna universal rt microrna pcr system, by Qiagen (1 mentions)
Quantifast sybr green pcr kit, by Qiagen (1 mentions)
Rotor gene 6000, by Qiagen (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
6 protocols using primescript 1st strand cdna kit
1
Quantitative RT-PCR for FCV Detection
2
Jurkat scFv Library Sequencing
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Following phenotypic screening and cell sorting, mRNA was isolated from 106–107 bulk Jurkat library cells using the Dynabeads mRNA DIRECT purification kit (Thermo Fisher Scientific, cat. 61,011) according to the manufacturer’s instructions. Subsequently, 1 µg of the resulting mRNA served as the template for cDNA synthesis using the PrimeScript 1st strand cDNA kit (Takara, cat. 6110B) and oligo-dT primers. The scFv sequences were amplified from the cDNA template using Phusion High-Fidelity PCR Master Mix with GC Buffer (NEB, cat. M0532S) and appropriate flanking primers situated upstream in the scFv signal peptide (Forward) and downstream in the CD28 extracellular spacer region (Reverse). Successfully amplified scFv pools were purified from 1% agarose gels using a ZymoCLEAN gel DNA recovery kit (Zymo Research, cat. D4002) and cloned into the pCHV101 phagemid vector using NcoI/SalI. E. coli TG1 cells were electroporated, and discrete ampicillin-resistant colonies were picked for high-throughput plate sequencing (Microsynth).
3
Genomic DNA and RNA Extraction
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Genomic DNA was extracted from leaves using a cetyl trimethylammonium bromide (CTAB) method [29 (link)]. Total RNA was isolated using RNAiso plus Reagent (Takara, Japan) according the manufacturer’s instructions and purified using DNase I. Complementary DNA was synthesized using a PrimeScript 1st Strand cDNA kit (Takara, Japan).
4
RT-qPCR Analysis of mRNA and miRNA
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Initially, 1 μg of total RNA was used for the synthesis of the first strand of cDNA using PrimeScript 1st strand cDNA Kit (TaKaRa Bio, Kusatsu, Shiga, Japan) and 500 ng of total RNA for the synthesis of the first strand micro cDNA using Universal cDNA Synthesis kit II, 8–64 rxns (Exiqon, Vedbæk, Denmark), respectively. qRT-PCR was achieved using QuantiFast SYBR Green PCR kit (QIAGEN, Hilden, Germany). The relative expression level of mRNAs and miRNAs were calculated using the 2-ΔCt method [45 (link)]. GAPDH (NCBI accession no. 2597) and β-ACTIN (NCBI accession no. 60), and miR-191-5p (miRBase accession no. MIMAT0000440 ) and U6 snRNP (NCBI accession no. 26827) were used as endogenous controls for normalizing of mRNA and miRNA expression levels, respectively. GAPDH and β-ACTIN were already reported to be used for the neurological studies [46 (link)], U6 is a universal reference for miRNA RT-qPCR studies [47 (link)], and miR-191-5p was also reported to be used for qRT-PCR of blood samples [48 ] and was introduced as a reliable endogenous control by the Exiqon company (miRCURY LNA Universal RT microRNA PCR system). Normalization was performed using geometric averaging [49 (link)] of miR-191-5p and U6 snRNP, GAPDH, and β-ACTIN expressions.
5
Comprehensive RNA Expression Analysis
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Cells were collected and preserved at −70°C until RNA extraction. Total RNA was isolated with QIAzol lysis reagent. Integrity and quality of RNA samples were checked using a Nanodrop (ND‐1000) spectrophotometer. 1 μg of the total RNA was subjected to reverse transcription using oligo‐dT and PrimeScript™ 1st strand cDNA kit (Takara, Japan). Transcript levels were determined using the SYBR Green master mix and Corbett Rotor‐Gene 6000. Gene expression level was normalized to the human GAPDH housekeeping gene. Relative quantification of gene expression relative to the positive control group was calculated using the ΔΔCt method. Primer sequences for qRT–PCR are listed in Table S1 .
6
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted using the RNeasy (Qiagen) method. First strand cDNA was generated using Prime Script 1 st Strand cDNA kit (Takara) according to manufacturer's instructions. Real time PCR was performed using CFX96 PCR system with SYBR Premix Ex Taq II kit (Takara).
The primers used for qRT-PCR analysis are listed in Table 2. Each PCR was carried out in at least three independent replicates. The p-values were estimated after comparing among the cell types using bilateral student test. In all analyses, 'ns' stands for not significant, for p-value> 0.05; '*' for p-value<0.05; '**' for p-value<0.01; '***' for p-value<0.001.
The primers used for qRT-PCR analysis are listed in Table 2. Each PCR was carried out in at least three independent replicates. The p-values were estimated after comparing among the cell types using bilateral student test. In all analyses, 'ns' stands for not significant, for p-value> 0.05; '*' for p-value<0.05; '**' for p-value<0.01; '***' for p-value<0.001.
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