To quantify mating-induced changes in gene expression, the middle midguts from 5 to 10 adult female flies were dissected. Total RNA was extracted using RNAiso Plus reagent (TaKaRa). cDNA was prepared with ReverTra Ace qPCR RT Master Mix with gDNA Remover (ToYoBo). Quantitative reverse transcription PCR (qRT-PCR) was performed using the Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 system (TaKaRa). Serial dilutions of a plasmid containing the open reading frame of each gene were used as standard. The amount of target RNA was normalized to ribosomal protein 49 (rp49) and then relative fold changes were calculated. The following primer pairs were used to measure transcript level: rp49 forward, 5ʹ-CGGATCGATATGCTAAGCTGT-3ʹ and reverse, 5ʹ-GCGCTTGTTCGATCCGTA-3ʹ; NPF forward, 5ʹ-CTCCGCGAAAGAACGATGTCAACAC-3ʹ and reverse, 5ʹ-CCTCAGGATATCCATCAGCGATCCG-3ʹ; NPFR forward, 5´-GATCCTGTCCAAGTACTGGCCCTAC-3´ and reverse, 5´-ACGATCACCTGATATCTGTCGAAGGC-3´. All qRT-PCR runs were performed in triplicate.
Universal sybr select master mix
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The Universal SYBR Select Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains SYBR Green I dye, which binds to double-stranded DNA, enabling the detection and quantification of target sequences. The master mix includes all the necessary components for qPCR, including DNA polymerase, dNTPs, and buffer.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus reagent, by Takara Bio (6 mentions)
Thermal cycler dice tp800 system, by Takara Bio (5 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Gdna remover, by Toyobo (2 mentions)
Revertra ace qpcr rt master mix, by Toyobo (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (2 mentions)
Thunderbird sybr qpcr mix, by Toyobo (2 mentions)
Revertra ace qpcr master mix, by Toyobo (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Lightcycler 96, by Roche (2 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Thermal cycler dice real time system 2, by Takara Bio (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nucleospin mirna, by Macherey-Nagel (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
17 protocols using universal sybr select master mix
1
Mating-induced Transcriptome Changes in Flies
2
Quantitative Real-Time PCR Protocol
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Quantitative real-time PCR was performed using SYBR Premix ExTaq II (Takara) and Universal SYBR Select Master Mix (Applied Biosystems) on a StepOnePlus Real-Time PCR System (Applied Biosystems) with the PCR conditions recommended by the supplier. All measurements were performed in duplicate, and the difference in the duplicate threshold cycle was less than one cycle in all samples tested. All experiments were repeated at least three times. The primers used for real-time PCR are summarized in Table 2 .
3
Quantitative Gene Expression Analysis of Wasp Venom
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To confirm if these putative venom genes are strongly expressed in the venom gland, we performed quantitative reverse transcription PCR (qRT-PCR) analysis of the top five genes with high FPKM values (Table 7 ). The venom glands and the other carcass from 20 wasp adults were dissected and total RNA was extracted using RNAiso Plus reagent (TaKaRa). cDNA was prepared with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative PCR was conducted using the Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 system (TaKaRa). For the relative quantification of expression, the A. japonica orthologue of Ribosomal protein L32 (RpL32-Aj) was identified by tBLASTn analysis using amino acid sequence of D. melanogaster RpL32. The target gene expression level was normalized to RpL32 and then relative fold changes were calculated by the delta-delta Ct method. The mean values were from three independent experiments. The primers used for qRT-PCR are described in Table 1 .
4
qRT-PCR Analysis of Gene Expression
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Total RNA from cells was purified by using NucleoSpin miRNA (MACHEREY-NAGEL). The concentration of total RNA was assessed by use of UV spectrophotometry. RNA integrity was checked with an Agilent 2100 Bioanalyzer (Agilent). cDNA was reverse transcribed from 0.5 μg of the total RNA by using a PrimeScript RT Reagent Kit (Takara). The cDNA was amplified with Universal SYBR Select Master Mix (Applied Biosystems), and signals were recorded with a Takara Thermal Cycler Dice Real Time System II. Relative expression levels were quantified by the ΔCt method and normalized to B2m. The gene used as an internal control was selected from Tbp, B2m, Gapdh, Actb, Hprt by using the NormFinder algorithm (https://moma.dk/normfinder-software ).
5
Quantifying Gene Expression in Drosophila
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To quantify the changes in gene expression, the midguts from 8 to 10 adult female flies, the fly abdomen carcass from ten adult female flies, and the heads from 20 adult female flies were dissected for each sample. For Akh mRNA level quantification, 6 whole bodys of adult female flies were sampled. Total RNA was extracted using RNAiso Plus reagent (TaKaRa). cDNA was prepared with ReverTra Ace qPCR RT Master Mix with gDNA Remover (ToYoBo). Quantitative reverse transcription PCR (RT-qPCR) was performed using the Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 system (TaKaRa). Serial dilutions of a plasmid containing the open reading frame of each gene were used as standard. The amount of target RNA was normalised to ribosomal protein 49 (rp49) and then relative fold changes were calculated. The primers used to measure transcript levels are represented in Supplementary Data 6 .
6
Gene Expression Changes in Mated Flies
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To quantify mating-induced changes in gene expression, the ovaries from 2 adult female flies (4-day-old virgin or mated females) were dissected. Total RNA was extracted using RNAiso Plus reagent (TaKaRa). cDNAs were prepared with ReverTra Ace qPCR RT Master Mix with gDNA remover (ToYoBo). qRT-PCR was performed using the Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 system (TaKaRa). Serial dilutions of a plasmid containing the ORF of each gene were used as a standard. The amount of target RNA was normalized to an endogenous control ribosomal protein 49 (rp49), and the relative fold change was calculated. The primers for quantifying nobo, nvd, sro, spo, phm, dib, sad and shd were used in the previous studies [64 (link)–66 (link)].
7
Quantitative Analysis of Ch25h Expression
8
Quantitative gene expression analysis
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RNA was isolated using the RNAiso Plus reagent (TaKaRa). Genomic DNA digestion and cDNA synthesis were performed using the ReverTra Ace qPCR RT Kit (TOYOBO). qRT-PCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) or Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 or TP870 system (TaKaRa). Serial dilutions of a plasmid containing the ORF of each gene were used as a standard. The expression levels of the target genes were normalized to an endogenous control ribosomal protein 49 (rp49) in the same sample. The primers for quantifying D. melanogaster ouib and E75A are described in S3 Table . Primers amplifying nvd, sro, spok, phm, dib, sad and rp49 were previously described [22 (link),55 (link)].
9
Quantitative RT-PCR for Arabidopsis
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Total RNA was isolated using the Trizol reagent (Invitrogen) essentially as described before (Kim et al. 2008 (link)). Total RNA (100 ng) was used for the first-strand cDNA synthesis by AMV reverse transcriptase (Nippon Gene). Quantitative PCR was performed using Universal SYBR Select Master Mix (Applied Biosystems) in a StepOne Real-Time PCR System (Applied Biosystems). The Arabidopsis tubulin gene (AtTub) was used as an internal control. Primer sets for each gene amplification were as follows: 5′-GAGGGAGCCATTGACAACATCTT-3′ and 5′-GCGAACAGTTCACAGCTATGTTCA-3′ (for AtTub), 5′-GCGCGTCGACGTTGACGTCGAGCACCAAC-3′ and 5′-CCATCGATTGGTCTCCTTTTGGAGGCC-3′ (for CMV).
10
Quantification of Gene Expression in Drosophila
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To quantify the changes in gene expression, the middle midguts from 8 to 10 adult female flies, the fly abdomen carcass from ten adult female flies, and the heads from 20 adult female flies were dissected for each sample. Total RNA was extracted using RNAiso Plus reagent (TaKaRa).
cDNA was prepared with ReverTra Ace qPCR RT Master Mix with gDNA Remover (ToYoBo).
Quantitative reverse transcription PCR (RT-qPCR) was performed using the Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 system (TaKaRa).
Serial dilutions of a plasmid containing the open reading frame of each gene were used as standard. The amount of target RNA was normalised to ribosomal protein 49 (rp49) and then relative fold changes were calculated. The primers used to measure transcript levels are represented in Extended Data Table 6.
cDNA was prepared with ReverTra Ace qPCR RT Master Mix with gDNA Remover (ToYoBo).
Quantitative reverse transcription PCR (RT-qPCR) was performed using the Universal SYBR Select Master Mix (Applied Biosystems) with a Thermal Cycler Dice TP800 system (TaKaRa).
Serial dilutions of a plasmid containing the open reading frame of each gene were used as standard. The amount of target RNA was normalised to ribosomal protein 49 (rp49) and then relative fold changes were calculated. The primers used to measure transcript levels are represented in Extended Data Table 6.
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