Total RNA (0.5 μg) was used to synthesise cDNA with a ReverTra Ace qPCR RT Master Mix and a gDNA Remover Kit (Toyobo Co. Ltd, Osaka, Japan) according to the manufacturer’s instructions. For semi-quantitative reverse transcription–polymerase chain reaction (RT-PCR), the synthesised cDNA was added to a mixture of 1 U of Taq DNA polymerase (Lucigen, Middleton, WI, USA) and gene-specific primers. This was amplified using the T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, IN, USA). PCR products were separated by electrophoresis on 1.5% agarose gels and detected under ultraviolet light (Bio-Rad, Hercules, CA, USA). Real-time RT–PCR analysis was performed on a LightCycler 480 Real-Time Detection System (Roche Diagnostics, Indianapolis, IN, USA) using 2 × LightCycler 480 SYBR-Green Master Mix (Roche Diagnostics). The sequences of human-specific primers used are: Id2 – forward, 5′-TGGACTCGCATCCCACTATT-3′ and reverse, 5′-ATTCAGAAGCCTGCAAGGAC-3′ NGFR (nerve growth factor receptor) – forward, 5′- CTGCTGTTGCTGCTTCTGGG-3′ reverse, 5′- GGCTCACACACGGTCTGGTT-3′ CK-4 – forward, 5′-AGGAGGTCACCATCAACCAG-3′ and reverse, 5′-ACCTTGTCGATGAAGGAGGC-3′.
Lightcycler 480 sybr green master mix
Manufactured by Roche
Sourced in Germany, Switzerland, United States, United Kingdom
The LightCycler 480 SYBR Green Master Mix is a ready-to-use reagent for real-time PCR amplification and detection using the SYBR Green I dye. It contains all the necessary components, including DNA polymerase, dNTPs, and SYBR Green I, to perform quantitative PCR analysis.
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Lab products found in correlation
Lightcycler 480, by Roche (24 mentions)
Trizol reagent, by Thermo Fisher Scientific (17 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (13 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Trizol, by Thermo Fisher Scientific (10 mentions)
Lightcycler 480 2, by Roche (8 mentions)
Lightcycler 480 system, by Roche (6 mentions)
Rneasy kit, by Qiagen (6 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (5 mentions)
Lightcycler 480 real time pcr system, by Roche (5 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
Rneasy micro kit, by Qiagen (4 mentions)
Reverse transcription kit, by Roche (4 mentions)
Qscript cdna supermix, by Quanta Biosciences (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Light cycler 480 software 1, by Roche (3 mentions)
Random primer, by Thermo Fisher Scientific (3 mentions)
Tripure isolation reagent, by Roche (2 mentions)
Glass bead, by Merck Group (2 mentions)
123 protocols using lightcycler 480 sybr green master mix
1
RT-qPCR Analysis of Gene Expression
2
Quantifying PSY Expression Levels
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Quantitative analysis of PSY expression levels was performed with a Roche LightCycler 480 system using the 2× LightCycler 480 SYBR Green master mix (Roche) and a three-step program. The primers were listed in Supplemental Table 1 . A total amount of 30 μg protein was used for western blot analysis and detected by either GFP or RFP antibodies (Biorbyt) at a dilution of 1:5000 or 1:10,000, respectively. Goat anti-mouse HRP (Biorbyt) was used as the secondary antibody at a dilution ratio of 1:10,000. The fluorescent signals were detected using a chemiluminescent gel imaging system (Amersham Imager 600).
3
Quantifying eGFP Expression in Transfected Cells
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eGFP transgene expression at the mRNA level was detected in stably transfected cells and analyzed by quantitative reverse transcription PCR (qRT-PCR). From 5 × 106 stably transfected cells, the total RNAs were isolated using TaKaRa RNAiso Reagent according to the manufacturer’s instructions (TaKaRa Company). RNA was converted to cDNA using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, UK). qRT-PCR was performed on a Light Cycler 480 system (Roche) using the Roche LightCycler® 480 SYBR Green Master Mix. The eGFP primers were as follows: 5'-CTACGTCCAGGAGCGCACCATCT-3' (forward), 5'-GTTCTTCTGCTTGTCGGCCATGATAT-3' (reverse). GAPDH was used as an internal reference, and the primer sequences were as follows: 5'-CGACCCCTTCATTGACCTC-3' (forward), 5'-CTCCACGACATACTCAGCACC-3' (reverse). The qPCR procedure consisted of 40 cycles using the manufacturer’s recommended parameters. Relative eGFP mRNA was calculated using the 2−∆∆ Ct method. All experiments were repeated three times.
4
Quantifying Spinal Cord C3aR1 Expression
5
Quantifying Dnmt1 Knockdown Efficacy
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We assessed the effectiveness of Dnmt1 knockdown, using qRT-PCR. We synthesized cDNA using 500 ng RNA with qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). Validated primers were used from Bewick et al. [8 (link)]. GAPDH was used as the endogenous reference gene. We used Roche LightCycler 480 SYBR Green Master Mix with a Roche LightCycler 480 (Roche Applied Science, Indianapolis, IN) with 3 technical replicates of 10 μL reactions as previously reported [11 (link)]. We used the ΔΔCT method to estimate differences of expression using eGFP samples as our comparison group [29 (link)]. We had 14 7-day eGFP, 14 7-day Dnmt1, 14 14-day eGFP, and 13 14-day Dnmt1 biological replicates.
6
Quantifying Dnmt1 Knockdown in Insect Ovaries
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To assess the effectiveness of posttranscriptional knockdown of Dnmt1, females were dissected 11 days post-injection. Ovaries were removed from each female, flash frozen in liquid nitrogen, and stored at − 80 °C until processing. Total RNA (and DNA) was extracted from a single ovary per female using a Qiagen Allprep DNA/RNA Mini Kit (Qiagen, Venlo, The Netherlands) per manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 500 ng RNA with qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD).
Expression level of Dnmt1 was quantified by quantitative real-time PCR (qRT-PCR). Primers were designed for Dnmt1 using the O. fasciatus genome as a Ref. [52 (link)]. Actin and GAPDH were used as endogenous reference genes. Primer sequences can be found in Additional file1 : Table S2. We used Roche LightCycler 480 SYBR Green Master Mix with a Roche LightCycler 480 (Roche Applied Science, Indianapolis, IN) for qRT-PCR. All samples were run with 3 technical replicates using 10 μL reactions using the manufacturer’s recommended protocol. Primer efficiency calculations, genomic contamination testing, and endogenous control gene selection were performed as described by [53 (link)]. We used the ΔΔCT method [54 (link)] to examine differences in expression between control and ds-dnmt1-1 injected females.
Expression level of Dnmt1 was quantified by quantitative real-time PCR (qRT-PCR). Primers were designed for Dnmt1 using the O. fasciatus genome as a Ref. [52 (link)]. Actin and GAPDH were used as endogenous reference genes. Primer sequences can be found in Additional file
7
Quantitative RT-PCR Analysis of ftsH Genes
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For each sample of grapevine, broad bean, E. variegatus and S. titanus, 1 µg of RNA was used for cDNA synthesis using Superscript Reverse Transcriptase III and random primers following the manufacturer’s recommendations (Invitrogen, Vilnius, Lithuania). The final pellet was resuspended in 20 µL of RNase-free water. Real-time RT-PCR was performed using 1 µL of cDNA template in 1× Light Cycler 480 SYBR Green Master Mix (Roche, Mannheim, Germany) with 0.1–1 µM each primer in a final volume of 20 µL (Table 2 ). The reaction conditions were as follows: 15 min at 95 °C, followed by 36 or 40 cycles of 20 s at 95 °C; 30 s at 56, 60, 62, or 64 °C; and 30 s at 68 °C (Table 2 ). The specific primers designed to specifically amplify each ftsH gene are presented in Table 2 with the corresponding amplification conditions. The efficiency of RT-PCR varies between 90% and 100%, and all primers used show a single melting peak temperature between 74.1 °C (ftsH3) and 78.4 °C (FD-gyrA).
The three biological replicates (plants or insects) were run in duplicate on each of the three experimental replicates (plates) comprising negative and positive controls. Amplification of the DNA dilution series was performed to calculate the amplification efficiencies for each gene assay.
The three biological replicates (plants or insects) were run in duplicate on each of the three experimental replicates (plates) comprising negative and positive controls. Amplification of the DNA dilution series was performed to calculate the amplification efficiencies for each gene assay.
8
Quantifying Cell-Specific Gene Expression
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Total RNA was extracted from CD4+ TCs, Møs, SMCs and ECs using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed to cDNA using a high-capacity cDNA reverse transcription kit (cat. no. 4368813; Thermo Fisher Scientific, Inc.) at 92˚C for 5 min according to the manufacturer's protocol. Subsequently, qPCR was performed using LightCycler® 480 SYBR-Green Master Mix (Roche Diagnostics). The following thermocycling conditions were used for the PCR: 35 cycles at 92˚C for 30 sec and 58˚C for 40 sec, and 72˚C for 35 sec. The primer pairs used for qPCR were validated in previous studies, and were as follows: TSP-2, forward: 5'-TGAGTTCCAGGGCACACCA-3'; reverse: 5'-GGCTTTCTGGGCAATGGTA-3'; Bax, forward: 5'-TTGCTGATGGCAACTTCAAT-3'; reverse: 5'-GATCAGCTCGGGCACTTTAG-3'; Bcl2, forward: 5'-CAGAAGATCATGCCGTCCTT-3'; reverse: 5'-CTTTCTGCTTTTTATTTCATGAGG-3' (24 (link),25 (link)). mRNA levels were normalized to the internal reference gene GAPDH levels using the 2-ΔΔCq method (26 (link)).
9
Quantitative Real-Time PCR Analysis
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cDNAs were diluted 5-fold and 2 μl of diluted cDNA were then used for each polymerase chain reaction (PCR) or quamtitative PCR reaction. All primers were designed using Primer3 with default parameters (see a list in Supplementary Table S1 ).
PCR reactions were performed using the Taq CORE kit (MP Biomedicals) according to the manufacturer protocol. A first cycle of 15 min at 95°C was followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 30s at 72°C. The reaction was ended with an extension step for 3 min at 72°C. Visualization and quantification of amplified products was performed with a LabChip HT DNA assay on an automated microfluidic station (25 (link)) (Caliper, Hopkinton, MA, USA). For all experiments, quantifications of PCR triplicates are shown as a mean of their percent spliced in (PSI) or percentage spliced exclusion (PSE). The ΔPSI value was obtained by subtracting the PSI value of SOX9 KD from that of the corresponding control.
Quantitative RT-PCR reactions were performed using LightCycler® 480 SYBR Green Master Mix and amplified on the LightCycler® 480 instrument (Roche) with primers listed inSupplemental Table S1 . Relative levels of gene expression were analyzed using the 2ΔΔCt method and compared to the expression of the human housekeeping gene MRPL19. All analyses were performed according to the MIQE guidelines (26 (link)).
PCR reactions were performed using the Taq CORE kit (MP Biomedicals) according to the manufacturer protocol. A first cycle of 15 min at 95°C was followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 30s at 72°C. The reaction was ended with an extension step for 3 min at 72°C. Visualization and quantification of amplified products was performed with a LabChip HT DNA assay on an automated microfluidic station (25 (link)) (Caliper, Hopkinton, MA, USA). For all experiments, quantifications of PCR triplicates are shown as a mean of their percent spliced in (PSI) or percentage spliced exclusion (PSE). The ΔPSI value was obtained by subtracting the PSI value of SOX9 KD from that of the corresponding control.
Quantitative RT-PCR reactions were performed using LightCycler® 480 SYBR Green Master Mix and amplified on the LightCycler® 480 instrument (Roche) with primers listed in
10
Quantitative RT-PCR Gene Expression Analysis
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Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) was carried out as described previously (Furuta et al., 2014 ) using a LightCycler 480 (Roche) with LightCycler 480 SYBR Green master mix (Roche) and the manufacturer's qRT‐PCR program recommendations. Four technical repeats were carried out to assess the gene expression levels. Gene expression was normalized to UBQ10, as described previously (Furuta et al., 2014 ).
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