Total RNAs were extracted from apple in vitro shoot cultures using TRIzol Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Two micrograms of total RNAs was used to synthesize first‐strand cDNA. For real‐time quantitative PCR, the specific primer pairs are given in Table S1. The reactions were performed using SYBR Green MasterMix (SYBR Premix EX Taq TM, Dalian, China), as described by the manufacturer. Real‐time quantitative PCRs were performed using a BIO‐RAD iQ5 (Hercules, CA) instrument. The expression values obtained were normalized against 18s rRNA by the cycle threshold (Ct) 2 − Δ Δ C t
Iq5 2
Manufactured by Bio-Rad
Sourced in United States
The IQ5 2.0 software is a real-time PCR data analysis software. It is designed to process and analyze data generated by Bio-Rad's real-time PCR systems.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand synthesis supermix kit, by Thermo Fisher Scientific (18 mentions)
Rneasy mini kit, by Qiagen (18 mentions)
Taqman mirna rt kit, by Thermo Fisher Scientific (4 mentions)
Taqman microrna assays kit, by Thermo Fisher Scientific (4 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Icycler iq5, by Bio-Rad (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taqtm, by Takara Bio (2 mentions)
Pcr instrument, by Bio-Rad (2 mentions)
Sybr green supermix kit, by Bio-Rad (2 mentions)
Rna nano chip, by Agilent Technologies (2 mentions)
Universal human probe library system, by Roche (2 mentions)
Goscript reverse transcription system, by Promega (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Primescript reverse transcription kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Prime scirpt reverse transcription kit, by Takara Bio (1 mentions)
32 protocols using iq5 2
1
RNA Extraction and qPCR for Apple Cultures
2
RT-qPCR Analysis of Gene Expression
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We used TRIzol (Invitrogen, USA) to lyse the cells. Then total RNA was extracted after the cells were treated accordingly. After initially removing genomic DNA from RNA, the RNA was purified using DNase I. The Prime Script Reverse Transcription Kit (TaKaRa, Otsu, Shiga, Japan) instructions were followed for reverse transcription of RNA, and RT-PCR was performed according to SYBR® Premix Ex TaqTM (TaKaRa, Japan) kit using the StepOne Plus Real-time PCR System [Applied Biosystems (AB), Foster City, CA, USA]. For each sample, 3 replicate wells were performed and repeated the assay twice. We analyzed the data using a Bio-Rad PCR instrument and software iQ5 2.0 (Bio-Rad Laboratories, Hercules, CA, USA). As internal parameters, we used the β-actin and U6 genes, and we calculated gene expression using the 2-ΔΔCt method.
3
Quantifying NLRP3 and Caspase-1 Expression
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The total RNA in both PCa cell lines (in vitro) and PCa samples (in vivo) was extracted using an RNeasy Mini Kit (ThermoFisher Scientific, USA) according to the manufacturer’s protocol. Each sample was lysed with 1 ml TRIzol. The initially extracted RNA was treated with DNase I to remove genomic DNA and repurify the RNA. With the help of the Prime Scirpt Reverse Transcription Kit (Takara) instructions, RNA reverse transcription was performed, real-time PCR was performed according to the SYBR® Premix Ex TaqTM (Takara) Kit instructions, and the PCR reaction was performed using the StepOne Plus Real-time PCR System (Applied Biosystems, Foster City, CA, USA) system. Each sample was repeated with 3 replicate wells repeated twice. Bio-Rad PCR instrument and software iQ5 2.0 were used to analyze and process the data. GAPDH was used as internal parameters, and NLRP3 and caspase-1 expression was calculated by 2−ΔΔCt method. The following primers were used for qRT-PCR reactions:
NLRP3:
forward, 5′-ATGTGGGGGAGAATGCCTTG-3′,
reverse, 5′-TTGTCTCCGAGAGTGTTGCC-3′;
caspase-1:
forward, 5′-GCAATGAAGACGAAGGCGAC-3′,
reverse, 5′-GTGCCCGTGCGAGATTTTAG-3′;
GAPDH:
forward, 5′-GAAGGTGAAGGTCGGAGTC-3′,
reverse, 5′-GAAGATGGTGATGGGATTTC-3′.
NLRP3:
forward, 5′-ATGTGGGGGAGAATGCCTTG-3′,
reverse, 5′-TTGTCTCCGAGAGTGTTGCC-3′;
caspase-1:
forward, 5′-GCAATGAAGACGAAGGCGAC-3′,
reverse, 5′-GTGCCCGTGCGAGATTTTAG-3′;
GAPDH:
forward, 5′-GAAGGTGAAGGTCGGAGTC-3′,
reverse, 5′-GAAGATGGTGATGGGATTTC-3′.
4
Quantitative Analysis of lncRNA and miRNA
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We extracted total RNA from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and purified by DNase I treatment. Extracted RNA was reversely transcribed into complementary deoxyribose nucleic acid (cDNA) using Primescript RT Reagent (TaKaRa, Otsu, Japan). The cDNA was amplified by real-time quantitative PCR using SYBR®Premix Ex Taq™ (Ta-KaRa, Otsu, Japan). Primer sequences were as follows: LINC00339, forward, 5′-GGTTGACGAAGTCTGGAA-CG-3′; reverse, 5′-GCCCATCATTTCATTGGGTA-3′; glyceraldheyde 3-phosphate dehydrogenase (GAPDH), forward, 5′-GGTGAAGGTCGGAGTCAACG-3′; reverse, 5′-CCATGTAGTTGAGGTCAATGAAG-3′; MiRNA-30a-5p, forward,5'-TACGGATCCCCTT-CATCTTACTTTTTTCCCCCAA-3'; reverse,5'-ATCGC-TAGCGAAACTAGAAGCTCGGTGATGAATA-3'; U6, forward, 5'-CGCTTCGGCAGCACATATAC-3'; reverse, 5'-TTCACGAATTTGCGTGTCAT-3'. Each sample was performed in triplicate, and analyzed by iQ5 2.0 (Bio-Rad, Hercules, CA, USA).
5
Quantifying DENV-2 Inhibition by Anisomycin and Ribavirin
6
Assessing Gene Expression in Plant Roots
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RNA was extracted from the roots of each of the four cultivars using the EASYspin Plus Plant RNA Kit (Aidlab Biotechnologies, Beijing, China) [60 ] according to the manufacturer’s instructions. The RNA was quantified using a BIOMATE 3 spectrophotometer (Thermo Scientific, Worcester, MA, USA) and its integrity was confirmed using 1% agarose gel electrophoresis. A total of 1 mg of RNA was reverse-transcribed into cDNA using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). A control amplicon was generated using the following primers for amplification of β-actin (SIN_1009011): forward primer, 5′-TTTGAGCAGGAACTGGACACT-3′, and reverse primer, 5′-ACAACACTTCTGGACAACGGA-3′. Gene expression levels were determined by performing qRT-PCR in triplicate on an Icycler iQ5 (Bio-Rad) using the SYBR Green Supermix kit (Bio-Rad), all according to the manufacturer’s instructions. Data were analyzed using iQ5 2.1 software (Bio-Rad) and the 2–ΔΔCT method [61 ].
7
Quantitative PCR for JUNV RNA Detection
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Total RNA was extracted using TRI reagent (Genbiotech; Antibes, France) and the detection of JUNV genomic S RNA by PCR was performed as previously described [31 (link)]. Briefly, cDNA was synthesized using the primer N (+) (5′-CGCACAGTGGATCCTAGGC-3′) complementary to positions 3393 to 3501 of the genomic S RNA. Quantitative PCR reaction was conducted using JUNV-specific primers N (−) (5′-GGCATCCTTCAGAACAT-3′) and N (+), and SYBR Green (Fast Start Universal SYBR Green Master, Roche; Basel, Switzerland), to generate a 186 bp amplification fragment comprising the 3′ end of the N coding sequence. The PCR cycle progression for N (+)/N (−) primers was as follows: 5 min at 95 °C and 45 cycles of 30 s at 95 °C, 20 s at 48 °C, 30 s at 72 °C followed by 10 s at 82 °C using the MyiQ™2 Two Color Real-Time PCR Detection System (BioRad; Hercules, CA, USA). The cellular gene actin was used as standard for normalization. Average viral RNA Ct values were normalized to the average Ct values of actin (ΔCt = Ct virus − Ct actin) and ΔΔCt (ΔCt untreated cultures − ΔCt drug-treated cultures) based fold change calculations were set using Bio-Rad iQ5 2.1 software.
8
Real-Time PCR Gene Expression Analysis
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Total RNA was isolated using the RNeasy Mini kit (QIAGEN), and cDNA was synthesized from equivalent total RNA using a SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen) according to the manufacturer's instructions. Primers used for amplification of target genes are displayed in Supplemental Table 1 . Amplification was carried out using an iCycler IQ Real-Time PCR Detection System, and cycle threshold (Ct) values were tabulated in duplicate for each gene of interest in each experiment. “No template” (water) controls were used to ensure minimal background contamination. Using mean Ct values tabulated for each gene, and paired Ct values for β-actin as a loading control, fold changes for experimental groups relative to assigned controls were calculated using automated iQ5 2.0 software (Bio-Rad).
9
RNA Isolation and qRT-PCR Analysis
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Total RNA was isolated using the RNeasy Mini kit (QIAGEN), and cDNA was synthesized from equivalent total RNA using a SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen) according to the manufacturer's instructions. Primers used for amplification of target genes are displayed in Supplemental Table 1 . Amplification was carried out using an iCycler IQ Real-Time PCR Detection System, and cycle threshold (Ct) values were tabulated in duplicate for each gene of interest in each experiment. “No template” (water) controls were used to ensure minimal background contamination. Using mean Ct values tabulated for each gene, and paired Ct values for β-actin as a loading control, fold changes for experimental groups relative to assigned controls were calculated using automated iQ5 2.0 software (Bio-rad).
10
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated using the RNeasy Mini kit (QIAGEN), and cDNA was synthesized from equivalent total RNA using a SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen) according to the manufacturer's instructions. Primers used for amplification of target genes are listed in Supplementary Table 2 . Amplification was carried out using an iCycler IQ Real-Time PCR Detection System, and cycle threshold (Ct) values were tabulated in duplicate for each gene of interest in each experiment. “No template” (water) controls were used to ensure minimal background contamination. Using mean Ct values tabulated for each gene, and paired Ct values for β-actin as a loading control, fold changes for experimental groups relative to assigned controls were calculated using automated iQ5 2.0 software (Bio-rad). For amplification of viral miRNAs, cDNA was synthesized using the Taqman miRNA RT kit (Applied Biosystems), and qPCR was performed using the Taqman MicroRNA Assays kit (Applied Biosystems) and a 7500 Real Time PCR System. Fold changes for microRNA were calculated using paired Ct values for RNU6B as recommended by the manufacturer (Applied Biosystems).
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