RT-PCR assays were conducted as described in our previous study (Liao et al. 2015 (link)). Total RNA from C. glutamicum cells was extracted during the exponential phase using an RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China), and the RNA concentration was determined by a microplate reader (BioTek Instruments, Winooski, VT, USA). The cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) and a Touch Real-Time PCR System (Bio-Rad Hercules, CA, USA), using the SYBR Premix Ex TaqTM II (TaKaRa, Shiga, Japan) on a Bio-Rad CFX96. cDNA (100 ng) was used as template. The PCR conditions were: 94 °C for 30 min, then 45 cycles at 94 °C for 5 s and 60 °C for 30 min. The data were normalized as per the 16S rRNA expression. The primers for RT-PCR are presented in the Additional file 1 .
Touch real time pcr system
Manufactured by Bio-Rad
Sourced in United States
The Touch Real-Time PCR System is a laboratory equipment used for performing real-time polymerase chain reaction (PCR) analysis. It is designed to amplify and quantify targeted DNA sequences in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Power sybr green pcr master mix, by Bio-Rad (3 mentions)
Taqman master mix, by Thermo Fisher Scientific (2 mentions)
C1000 touch pcr system, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Sybr premix ex taqtm 2, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Rna extraction kit, by Tiangen Biotech (1 mentions)
Rt premix, by Bioneer (1 mentions)
Oligo dt18, by Bioneer (1 mentions)
Qpcr master mix, by Bioneer (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rapidout dna removal kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Iqtm sybr green supermix, by Bio-Rad (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
6 protocols using touch real time pcr system
1
RT-PCR Assay for C. glutamicum Transcripts
2
Quantitative RT-PCR Viral Gene Expression
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Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and samples (500 ng) mixed with RT-PreMix (Bioneer, Daejeon, Korea) and OligodT18 (Bioneer, Daejeon, Korea) to synthesize cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using the Touch Real-Time PCR System (Bio-Rad, Hercules, CA) with qPCR Master Mix (Bioneer, Daejeon, Korea) and the following primers: HA (5′-ttgctaaaacccggagacac-3′ and 5′-cctgacgtatttgggcact-3′); NP (5′-gaatggtgctctctgcttttga-3′ and 5′-tccactttccgtttactctcctg-3′); PA (5′-aagtgccataggccaggtttc-3′ and 5′-cctcatctccattccccatttc-3′); M2 (5′-gaaaggagggccttctacgg-3′ and 5′-tcgtcagcatccacagcac-3′); canine IFN-β (5′-ccagttccagaaggaggaca-3′ and 5′-tgtcccaggtgaagttttcc-3′); canine Mx1 (5′-gaatcctgtacccaatcatgtg-3′ and 5′-taccttctcctcatattggct-3′); and canine β-actin (5′-tgccttgaagttggaaaacg-3′ and 5′-ctggggcctaatgttctcaca-3′) (Shoji et al., 2015 (link)). β-Actin expression was used as an internal reference, and relative expression of each gene was calculated using the ΔΔCt method. Every reaction was performed in triplicate.
3
Quantitative real-time PCR analysis of gene expression
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Total RNA from mouse samples was extracted with TRIzol™ reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RNA quality and concentrations were measured by Nanodrop Spectrophotometer ND1000 (Nanodrop Technologies, Inc., Wilmington, NC, USA). RNA (1–5 µg) was then incubated with DNase I (RapidOut DNA Removal kit, Thermo Fisher Scientific) for 30 min at 37 °C and reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Waltham, MA, USA, Thermo Fisher Scientific) according to the manufacturer’s instructions in a Touch PCR system (C1000, BIO-RAD, Hercules, CA, USA). Real-time PCR was performed using the Touch Real-Time PCR System (CFX384, BIO-RAD). The expression of genes was determined using Power SYBR Green PCR Master Mix (BIO-RAD) or TaqMan master mix (Applied Biosystems). SYBR Green primers were tested with Primer-Blast software (National Center for Biotechnology Information, Bethesda, MD, USA; https://www.ncbi.nlm.nih.gov/tools/primer-blast , accessed on 13 January 2023). Primer sequences are shown in Table 2 . For relative quantitation of gene expression, the comparative Ct method was used [2−ΔΔCt, where ΔCt represents the difference in threshold cycle between the target and a housekeeping gene (36b4)] [60 (link)].
4
Quantitative RT-PCR for Gene Expression
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RNA from mouse and human samples was extracted with TRIzol™ reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. RNA quality and concentrations were measured by Nanodrop Spectrophotometer ND1000 (Nanodrop Technologies, Inc. Wilmington, NC, USA). RNA (1–5 µg) was then incubated with DNase I (RapidOut DNA Removal kit, Thermo Fisher Scientific) for 30 min at 37 °C and reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific) according to the manufacturer’s instructions in a Touch PCR system (C1000, BIO-RAD, Hercules, CA, USA). Real-time PCR was performed using the Touch Real-Time PCR System (CFX384, BIO-RAD). The expression of genes was determined using Power SYBR Green PCR Master Mix (BIO-RAD) or TaqMan master mix (Applied Biosystems). SYBR Green primers were obtained from published studies and tested with Primer-Blast software (National Center for Biotechnology Information, Bethesda, MD, USA; https://www.ncbi.nlm.nih.gov/tools/primer-blast ). Primer sequences are shown in Table 4 . 36b4 and β-ACTIN were used as housekeeping genes in the mouse and human study respectively. Relative expression of the specific genes was determined using the 2−∆∆Ct method [88 (link)].
5
Transcriptional Analysis of Plant Responses
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Frozen cotyledons (7 and 11 dpi) were ground in liquid nitrogen with pestles and mortars. The total RNA was extracted with TRI reagent (Sigma-Aldrich) (St. Louis, MO USA). According to the manual, the total RNA was purified by DNaseI treatment with a recombinant DNaseI, RNase-free kit (Roche). Purified RNA was used to synthesize cDNA by employing the GOScript Reverse Transcription System (Promega). The cDNA stock solution was diluted to 100 ng/µL. Quantitative-PCR was performed by loading 1 µL of cDNA (100 ng) into the 10 µL reaction system of the IQTM SYBR® Green Supermix (BioRad, Hercules, CA, USA). The experiments were based on three biological replicates. The RT-qPCR experiments were run by Touch Real-Time PCR System (BioRad, Hercules, CA, USA).
The qPCR program used for all of the analysed genes (except for COI1 and ACO1) was 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s and 60 °C for 20 s. This was followed by a melting curve analysis.
All qPCR primers are compiled inSupplementary Table S1 . The relative level of gene expression was analysed with the 2-ΔΔCT method described by Livak and Schmittgen, (2001) [32 (link)]. Actin was used as a reference gene to normalize the expression of the target genes.
The qPCR program used for all of the analysed genes (except for COI1 and ACO1) was 95 °C for 3 min, followed by 39 cycles of 95 °C for 15 s and 60 °C for 20 s. This was followed by a melting curve analysis.
All qPCR primers are compiled in
6
Quantifying Adipocyte Gene Expression
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Total RNA was isolated from fully differentiated brown adipocytes and hMSC-derived adipocytes, as well as from iWAT and iBAT of mice, and then reverse transcribed to cDNA as previously described [34 (link)]. mRNA levels were determined using predesigned Taqman® Assays-on-Demand (Applied Biosystems, Waltham, MA, USA) or analyzed by Power SYBR Green PCR Master Mix (Bio-Rad) using the Touch Real-Time PCR System (CFX384, Bio-Rad). Cyclophilin A (Ppia), 36B4 and 18S were used as housekeeping genes (see Tables S1 and S2 for primers information). Relative expression of the genes was calculated by the 2−ΔΔCt method [38 (link)].
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