qRT-PCR was carried out to validate the quality of RNA-Seq data. Total RNA was extracted as described above. Each RNA sample was treated with RNase-free DNase I (TaKaRa, Shiga, Japan) following the manufacturer's protocol to remove any residual genomic DNA. DNase I-treated RNA (2 mg) was subjected to reverse transcriptase using oligo (dT) primer and PrimeScriptTM Reverse Transcriptase (TaKaRa, Shiga, Japan) according to the manufacturer's protocol. Total RNA (2 μg) was used to synthesize cDNA with PrimeScript™ RT reagent Kit (Perfect Real Time, TaKaRa, Japan). The C. militaris housekeeping gene, glyceraldehyde-phosphate dehydrogenase (Cmgapdh), was used as an internal control for normalization. Primers for the qRT-PCR of 51 DEGs were designed with Premier 6.0 software and are shown in S1 Table . Three biological replicates were performed per sample.
Primescript reverse transcriptase
Manufactured by Takara Bio
Sourced in Japan, China, United States, Germany, France
PrimeScript Reverse Transcriptase is a lab equipment product designed for the reverse transcription of RNA into cDNA. It is a recombinant reverse transcriptase enzyme that catalyzes the conversion of single-stranded RNA into double-stranded cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (125 mentions)
Sybr premix ex taq 2, by Takara Bio (52 mentions)
Trizol, by Thermo Fisher Scientific (42 mentions)
Rneasy mini kit, by Qiagen (34 mentions)
Sybr premix ex taq, by Takara Bio (27 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (21 mentions)
Cfx connect real time system, by Bio-Rad (17 mentions)
Rnaiso plus, by Takara Bio (16 mentions)
Trizol reagent, by Takara Bio (15 mentions)
Sybr premix ex taqtm 2, by Takara Bio (14 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (12 mentions)
Lightcycler 480, by Roche (12 mentions)
Thunderbird sybr qpcr mix, by Toyobo (11 mentions)
Rnaiso plus reagent, by Takara Bio (11 mentions)
Dnase 1, by Takara Bio (10 mentions)
Lightcycler 96, by Roche (10 mentions)
Dnase 1, by Thermo Fisher Scientific (10 mentions)
Trizol, by Takara Bio (10 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (9 mentions)
Rneasy plus mini kit, by Qiagen (8 mentions)
577 protocols using primescript reverse transcriptase
1
qRT-PCR Validation of RNA-Seq Data
2
Quantification of Islet-like Cell Transcripts
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On day 0 and day 6 of islet-like cell differentiation, cell aggregates were collected with TRIZol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was isolated by adding chloroform (Wako) with centrifugation of 15,000× g for 15 min. The 2-propanol (Wako) was added to collected supernatants and centrifuged at 15,000× g for 15 min to make a purified RNA pellet. The RNA pellet was washed with 75% ethanol and dissolved in RNase-free water. The concentration of RNA was measured by NanoDrop (Shimadzu, Kyoto, Japan) and 100 ng of RNA from each sample was reverse–transcribed by Prime ScriptTM Reverse Transcriptase (Takara Bio Inc., Kusatsu, Japan). After reverse transcription, transcribed complementary DNA samples were quantitated by SYBR Green gene expression assays and detected by StepOne Plus (Thermo Fisher Scientific). Target genes primer sequences are shown in Supplementary Table S1 .
3
RNA Extraction and qRT-PCR Analysis in INS-1 Cells
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Total RNA from INS-1 cells was extracted with RNAiso Plus solution (Takara, Japan). Then, 1 ug RNA was reverse-transcribed into cDNA using PrimeScriptTM Reverse Transcriptase (Takara, Japan). Real-time PCR was conducted with the SYBR Green PCR kit (Takara, Japan). Relative expression levels of target mRNAs were normalized to β-actin and were calculated based on the 2-ΔΔCt comparative method. Primer sequences are listed in Table 2
4
Kinetics of IBV infection in Vero cells
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Vero cells were seeded on 35-mm dishes and infected with 1 MOI IBV when the cell monolayer was approximately 90% confluent. The infected cells were treated with MβCD or Mevastatin for 1 h at 4°C and harvested at certain time points [0, 8, 12, 18, and 24 h post-infection (hpi)]. Total RNA was extracted, and the cDNA template was synthesized with PrimeScriptTM Reverse Transcriptase (Takara, Dalian, China). The target gene was amplified by PCR with synthesized cDNA and specific primer pairs. The primers used for amplification are listed in Table 1 . The amplification program was set at 94°C for 25 s, 56°C for 25 s, and 72°C for 20 s for 20 cycles. The sizes and specificity of the PCR products were verified by agarose gel electrophoresis. The relative expression levels were calculated by the 2-ΔΔCT method against the uninfected controls. GADPH gene was set as the endogenous control.
5
Gene Expression Analysis via qPCR
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Total RNA was harvested from synchronous populations, using TRIzol (Invitrogen) for lysing. For cDNA synthesis, mRNA was reverse transcribed using a iScriptTM cDNA Synthesis Kit (BioRad) and PrimeScriptTM Reverse Transcriptase (Takara). Quantitative PCR was performed in triplicate using a Bio-Rad CFX96 Real-Time PCR system (Bio-Rad). The following set of primers was used for paqr-1: 5′-ACAGCACAACTGTACAGGTGAAA-3′ and 5′-TCCCTTTTTACGACGATATCTAAGA-3′ and results were normalized to genomic DNA using the following primers for pmp-3: 5′-ATGATAAATCAGCGTCCCGAC-3′ and 5′-TTGCAACGAGAGCAACTGAAC-3′.
6
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted using RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Genomic DNA (gDNA) was eliminated by digesting with RNase-free genomic DNA eraser buffer (Qiagen), and cDNA was obtained by reverse transcription of RNA using PrimeScriptTM reverse transcriptase (Takara). Power SYBR Green Master Mix (Takara) was used on a Roche 480 PCR system for qRT-PCR analysis. The qRT-PCR reactions were performed in triplicate for gene specific primers. The mRNA level was calculated by normalizing to the endogenous mRNA level of actin (internal control) using Microsoft Excel. Primer sequences are shown (Supplementary Table 1 , Supporting Information).
7
RNA-seq Library Preparation and Sequencing
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RNA samples for sequencing were isolated using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Total RNA was subjected to a reverse transcription reaction with poly (dT) primers using PrimeScriptTM reverse transcriptase (Takara, Shiga, Japan) according to the manufacturer's protocol. The library quality and quantity were measured using a Nanodrop spectrophotometer (ND-1000; Thermo Scientific, MA, USA). Paired-end reads were sequenced using the NovaSeq-6000 platform (Illumina, CA, USA). Reference genome sequence data from Homo sapiens were obtained from the Ensemble genome browser (assembly ID: GRCh38). Reference genome indexing and read mapping of samples were performed using STAR software (ver. 2.6.1a).20 (link) FeatureCounts (ver. 1.6.2) software was then used to calculate the generated binary alignment map files.21 (link) The RNA-seq dataset generated in this study is available in the GEO public database under data series accession number GSE186043.
8
Quantitative RNA Extraction and qRT-PCR Analysis
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The NucleoSpin RNA plant kit (Macherey-Nagel) was used to extract total RNA from 100 mg of leaves. RNA concentrations were measured on a NanoDrop2000 (Thermo Scientific, USA). Quantitative RT-PCR (qRT-PCR) was performed with cDNA synthesized with the Prime ScriptTM Reverse Transcriptase (Takara) from 500 ng of total RNA. Design of the specific primers was done for each gene (see supplemental Table 1 ) with the Primer3plus software (www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi ). qRT-PCR was carried out using the LightCycler 480 SYBR Green I Master (Roche) in the quantitative PCR thermal cycler (LightCycler 480 real-time PCR system; Roche). Reaction preparation (total volume of 5 μl): 2 μl of cDNA diluted 10-fold, 2 μl of SYBR Green I Master, and 1 μM forward and reverse primers. The amplification profile was 95 °C for 10 min and 45 cycles (95 °C/15 s, 60 °C or 56 °C (depending on the gene)/15 s, and 72 °C/15 s). Reactions were performed in triplicates. Normalization of gene expressions was done using ACTIN2 as housekeeping gene. At least three biological replicates were performed for each gene tested.
9
Polysaccharide-Polyphenol RNA Extraction
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Total RNA was extracted from leaves via the centrifuging column method using the Tiangen polysaccharide polyphenol plant total RNA extraction kit, following the manufacturer’s protocol. Total RNA was dissolved in 30 µL of RNase-free water. Total RNA was quantified via spectrometry, and quality was checked on denatured agarose gels. First-strand cDNA was synthesized using PrimeScript TM Reverse Transcriptase (Takara, Dalian, China) following the manufacturer’s instructions. Total RNA with 10 mM dNTP in a total volume of 20 µL by incubating for 5 min at 65 °C, 1 h at 50 °C, and 5 min at 85 °C in accordance with the manufacturer’s instructions. First-strand cDNA was stored at −20 °C before use.
10
Soil RNA Extraction and cDNA Synthesis
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RNA was extracted from 2 g of soil samples stored at –80°C using an RNeasy PowerSoil Total RNA Kit (QIAGEN) in accordance with the manufacturer’s protocol, and 50 μL of resuspended RNA in RNase/DNase-free water was obtained. Extracted RNA was treated with DNase using a TURBO DNA-freeTM kit (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol and 60 μL of DNA-free soil RNA was obtained. cDNA synthesis from DNA-free soil RNA was conducted using PrimeScriptTM Reverse Transcriptase (Takara Bio) with random primers (hexadeoxyribonucleotide mixture; Takara Bio) in accordance with the manufacturer’s protocol, except for the addition of 1 mM dithiothreitol. DNA-free soil RNA was used at a 1/10 volume of the mixture.
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