For qRT-PCR analysis, mycelia of all tested strains were collected from PDA, WKM, and YES cultures for total RNA isolation with TRIzol reagent (Biomarker Technologies, Beijing, China). qRT-PCR was performed with Piko real-time PCR system (Thermo Fisher Scientific, Finland) by using the qPCR SuperMix (TransGen Biotech, Beijing, China). All utilized qRT-PCR primers were listed in Table 2 . The relative quantification of expression level for each gene was calculated following the 2−ΔΔCt method, and the expression of actin was used as internal control. Each sample for qRT-PCR assays was conducted with technical triplicates, and the experiment was repeated three times.
Piko real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Piko Real-Time PCR system is a compact and efficient instrument designed for real-time PCR analysis. It features a thermal cycler with a Peltier-based temperature control system and a sensitive fluorescence detection system to accurately measure and quantify nucleic acid targets in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Qpcr supermix, by Transgene (1 mentions)
Sensifast sybr kit, by Meridian Bioscience (1 mentions)
Pikoreal software 2, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using piko real time pcr system
1
Quantitative Real-Time PCR Analysis
2
Quantitative HCV Detection via Piko-Real-Time PCR
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HCV quantitation was performed on a Piko-Real-Time PCR System (Thermo-Scientific) using sensifast SYBR kit (Bioline) according to the manufacturer procedure.
3
qRT-PCR Gene Expression Analysis in S. solfataricus
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Primers for qRT-PCR were designed using Primer3 software version 4.1.0 and the annotated genome of S. solfataricus P2.
Gene expression was analyzed with a 96-well PikoReal-Time PCR System (Thermo Scientific, Waltham, MA, USA). Five µL of KAPA SYBR® FAST 2X (Sigma-Aldrich®, Merk KGaA, Burlington, USA) was used, along with 0.2 µL of each primer, and 0.5 µL of a 1:20 dilution of the cDNA.
Efficiency of each pair of primers was calculated from the average slope of a linear regression curve, constructed from qPCRs using a 10-fold dilution series (10 pg–10 ng) of S. solfataricus M16 chromosomal DNA as template. Cq values (quantification cycle) after 40 cycles were automatically determined by PikoReal software 2.1 (Thermo Scientific). Cq values of each transcript of interest was standardized to Cq value of housekeeping genes 16s rRNA and Rps2P. At least 3 biological replicates of each condition, and 2 technical replicates per qPCR reaction, were performed.
Gene expression was analyzed with a 96-well PikoReal-Time PCR System (Thermo Scientific, Waltham, MA, USA). Five µL of KAPA SYBR® FAST 2X (Sigma-Aldrich®, Merk KGaA, Burlington, USA) was used, along with 0.2 µL of each primer, and 0.5 µL of a 1:20 dilution of the cDNA.
Efficiency of each pair of primers was calculated from the average slope of a linear regression curve, constructed from qPCRs using a 10-fold dilution series (10 pg–10 ng) of S. solfataricus M16 chromosomal DNA as template. Cq values (quantification cycle) after 40 cycles were automatically determined by PikoReal software 2.1 (Thermo Scientific). Cq values of each transcript of interest was standardized to Cq value of housekeeping genes 16s rRNA and Rps2P. At least 3 biological replicates of each condition, and 2 technical replicates per qPCR reaction, were performed.
4
Quantitative Analysis of Tumor RNA
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Total RNA was extracted from tumor tissue using the RNAiso plus kit (TaKaRa, Japan). cDNAs were synthesized using the PrimeScript RT reagent kit (Takara, Japan). Quantitative PCR was performed using SYBR Premix Ex Taq (Takara, Japan) on Piko Real-Time PCR system (Thermo Scientific, Waltham, MA). Statistical analysis was conducted by GraphPad Prism five Software, Inc. (La Jolla, CA).
5
Quantitative RT-PCR in SH-SY5Y Cells
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SH-SY5Y wt cells were incubated as mentioned above and TRIzol reagent (Invitrogen) was used to isolate total RNA, of which 2 µg were used in reverse transcription with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Darmstadt, Germany). Fast SYBR Green Master Mix (Applied Biosystems, Darmstadt, Germany) was utilized for quantitative real-time polymerase chain reaction on a Piko Real-Time PCR System (Thermo Scientific, Waltham, MA, USA). Experiments were carried out according to the manufacturers’ protocols. Data evaluation and primer sequences are described in detail in [42 (link)].
6
qRT-PCR Analysis of M. oleifera Leaves
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The amplification of the cDNA in qRT-PCR was analyzed using a Thermo scientific Piko Real-Time PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cDNA for each sample was added in the appropriate amount for the amplification reaction. The PCR analysis was conducted using Piko Real-Time PCR according to the manufacturer’s instructions [36 ]. Three samples of the leaves of M. oleifera were prepared as technical replicates having one reference gene or internal control of 10 pico mole/µL primers (primer sequences are given in Table 2 ). The SYBR green dye 12.5 µL and 1.6 µL primer mixture (forward + reverse) were mixed and added in each well of the PCR plate. In addition, 8.9 µL of nuclease-free water and 2 µL cDNA were added in each well of the PCR plate and mixed gently. The denaturation temperature of 95 °C was set for 15 s. The annealing and extension both were carried out at 60 °C, which is the optimal temperature for the amplification by Taq DNA polymerase but at the same time ensures more efficient cleavage of the probe.
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