In order to determine the abundances of the nifH gene, a quantitative real-time PCR (qPCR) assay was performed using primer pairs 338F/518R as an internal control, and PolF/PolR as a quantification primer pair [49 (link)]. About 50 ng DNA, 1 μL primer pairs (5 pM), and 10 μL of 2 × SYBR Green mixture (Roche, Switzerland) were present in the final reaction volume of 20 μL. Each DNA sample had three technical duplicates and was incubated for 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C [25 (link)].
Sybr green mixture
Manufactured by Roche
Sourced in Switzerland, France, United States
SYBR Green mixture is a laboratory reagent used in real-time PCR (Polymerase Chain Reaction) assays. It is a fluorescent dye that binds to double-stranded DNA, allowing for the detection and quantification of amplified DNA sequences during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 2, by Roche (6 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Cfx real time pcr instrument, by Bio-Rad (4 mentions)
Trizol reagent, by Merck Group (2 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (2 mentions)
Superscript 4, by Thermo Fisher Scientific (2 mentions)
Lightcyclerr 480 2, by Roche (2 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Oligo dt primer, by Promega (2 mentions)
H3k27me3, by Merck Group (2 mentions)
Anti h3k9ac, by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Lightcycler system, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Rneasy purification kit, by Qiagen (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Qrt pcr, by Bio-Rad (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using sybr green mixture
1
Quantitative Analysis of nifH Gene
2
Quantification of nifH Gene Abundance
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To compare the relative abundance of the nifH gene, a quantitative real‐time PCR (qPCR) assay was performed. The primer pair of PolF–PolR was used for the quantification of the nifH gene and the primer pair of 338F − 518R for the 16 S rRNA was used as an internal control (Poly et al., 2001 (link); Yang et al., 2012 ). The final 20 μl reaction volume contains 50 ng DNA (∼1 μl), 1 μl primer pair (5 pM) and 10 μl of 2× SYBR Green mixture (Roche, Switzerland). Each group contained three biological replicates (i.e., three DNA samples), and each DNA sample contained three technical duplicates. The PCR program contained 95°C for 10 min, followed by 40 cycles consisting of 95°C for 15 s and 60°C for 1 min (Wen et al., 2019 (link)).
3
Quantifying CUG RNA Expression in Drosophila
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Total RNA was extracted from ten flies per genotype using Trizol reagent (Sigma). DNase I treatment and reverse transcription were performed as previously reported (Llamusi et al., 2013 (link)). To quantify the expression of CUG RNA, the common SV40 terminator in the pUAST vector was used as target of the primers (F: 5′-GGAAAGTCCTTGGGGTCTTC-3′, R: 5′-GGAACTGATGAATGGGAGCA-3′). Expression levels were normalized to the reference gene rp49 (F: 5′-ATGACCATCCGCCCAGCATAC-3′, R: 5′-ATGTGGCGGGTGCGCTTGTTC-3′) using the SYBR® Green mixture (Roche) under 2−ΔΔCt method. For each genotype, three biological samples were used and three technical replicates were performed.
4
Quantification of Host Gene Expression
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Total RNA was extracted from LX-2 cells using RNeasy Plus Mini Kit (QIAGEN, Venlo, Netherlands) according to the manufacturer’s instructions. Total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. RT-PCR testing was performed using the LightCycler system (Roche Diagnostics, Mannheim, Germany) and 5 μL of SYBR Green mixture (Roche), 2 μL of purified cDNA, and 1 μL of forward and reverse primer were used for PCR amplification. The primer sequences used were GAPDH (forward primer: 5’-agccacatcgctcagacac-3’ , reverse primer: 5’-gcccaatacgaccaaatcc-3’ ), IL-1β (forward primer: 5’-GCCAGTGAAATGATGGCTTAT-3’ , reverse primer: 5’-ggtcctggaaggagcactt -3’ ), and PKR (forward primer: 5’-AGCACACTCGCTTCTGAATC-3’ , reverse primer: 5’-CTGGTCTCAGGATCATAATC-3’ ). Relative mRNA expression levels of target host genes were defined by dividing their values by the amount of GAPDH mRNA, which were then evaluated by statistical analysis.
5
Quantitative RT-PCR for Gene Expression
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Total RNA was prepared from cultured cells or mouse tissues using RNeasy purification kit (Qiagen). One microgram RNA of each sample was reverse transcribed with a cDNA reverse transcription kit (Invitrogen) and subjected to Q-PCR by using SYBR green mixture (Roche) with Roche Light-Cycler Q-PCR System. Primers were purchased from Qiagen.
6
Allelic Expression Analysis by RT-qPCR
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After treatment with RNase-free DNase I (Life Technologies, 180868-015), first-strand cDNA was generated by reverse transcription with Superscript-IV (Life Technologies, 18090050) using random primers and 500 ng of RNA. Then, cDNA was amplified by real-time PCR with the SYBR Green mixture (Roche) using a LightCycler R 480II (Roche) apparatus. The relative expression level was quantified with the 2-delta Ct method that gives the fold change variation in gene expression normalized to the geometrical mean of the expression of the housekeeping genes Gapdh, Tbp, and Gus. The primer sequences are in Table S2 .
For allelic analysis, for each locus of interest, the parental allele origin of expression was assigned following direct sequencing of the cognate RT-PCR product that encompassed a strain-specific SNP (SNP details inTable S2 ).
For allelic analysis, for each locus of interest, the parental allele origin of expression was assigned following direct sequencing of the cognate RT-PCR product that encompassed a strain-specific SNP (SNP details in
7
Quantitative Analysis of miR-766 and PDCD5
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Total RNA was extracted from tissues and cells by using TRIZOL (Invitrogen, USA). Then, total RNA was reverse-transcribed into cDNA by using Reverse Transcription Kit (ThermoScientific, USA). qRT-PCR (Bio-Rad, USA) was performed by using SYBR Green Mixture (Roche, Switzerland). Primers used for qRT-PCR were shown as follows: miR-766 F: 5′-GCGGCCGCTATACACAGAGGATTGCTTAG-3′, R: 5′-ACGCGTCAGGCAACAGATTTC-3′; U6 F: 5′-CTCGCTTCGGCAGCACATATACT-3′, R: 5′-ACGCTTCACGAATTTGCGTGTC-3′; PDCD5 F: 5′-CAACAGGAAGCAAAGCAC-3′, R: 5′-GATCTTAACTTCTGCCTAGAC-3′; GAPDH F: 5′-GACGGCCGCATCTTCTTGT-3′, R: 5′-CACACCGACCTTCACCATTTT-3′. The primers were synthetized by Sangon Co., Ltd. (Shanghai, China). The relative expression of miR-766 and PDCD5 was normalized to U6 and GAPDH, respectively, and calculated according to the 2−ΔΔCt method.
8
Quantitative RT-PCR for Gene Expression
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siRNA transfections were carried out with Lipofectamine 2000 (Invitrogen, USA) and all plasmid transfections were performed with Polyethylenimine (PEI, Bender, Austria) according to the manufacturer's instructions. Total RNA was isolated using the Trizol reagent kit (Invitrogen, USA). cDNA was reverse-transcribed from the total RNA using the First Strand Reverse Transcription Kit (Fermentas, K1691). qRT-PCR was performed on an ABI 7500 Real time PCR system using a SYBR Green Mixture (Roche, 4913914001).
9
RT-PCR Analysis of Seedling Genes
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The RT and RT-PCR were performed as described previously (39 (link)). Leaves of seedlings at 10 days old were used for RNA isolation, and oligo(dT) primers (Promega) were used for RT. The CFX real-time PCR machine (Bio-Rad) and SYBR Green mixture (Roche) were used for RT-PCR. The relative expression of the genes was measured relative to UBIQUITIN 10 or the input. Data file S1 lists the primer sequences.
10
Real-Time PCR of Reverse Transcribed RNA
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RNA was reverse transcribed as described before and amplified by real-time PCR with a SYBR Green mixture (Roche) using a LightCycler® 480II (Roche) apparatus. For each condition, analyses were repeated four times, each in duplicate. The primer sequences are in Supplementary Tables S1 and S2.
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