Total RNA was extracted from human fibroblasts by using Tri reagent (Sigma, Saint Quentin Fallavier, France). The RNA pellet was resuspended in 25 μl of RNase-free distilled water and stored at −80 °C. The RNA was used for reverse transcription using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Charbonnieres, France) according to the manufacturer’s instructions. The reaction was carried out using 1 μg total RNA as template for the normalization of viral RNA to the amount of total RNA. The MaximaTM Probe/ROX qPCR Master Mix (2x) (Thermo Scientific) was used in qPCR experiment. Each reaction of 25 μL contained 400 nM of each primer, 200 nM of specific probe and 1x Maxima Probe/ROX qPCR Master Mix. Primers and probes sequences are listed in Supplementary Table S1 . The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). Viral RNA was quantified by comparing the sample’s threshold cycle (Ct) values with each virus RNA standard curve which was obtained as previously described47 (link), 54 (link).
Maximatm probe rox qpcr master mix 2x
Manufactured by Thermo Fisher Scientific
Sourced in France
The Maxima Probe/ROX qPCR Master Mix (2x) is a ready-to-use solution for real-time quantitative PCR (qPCR) experiments. It contains all the necessary components, including a hot-start Taq DNA polymerase, dNTPs, and buffer, to perform qPCR reactions with probe-based detection methods.
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3 protocols using maximatm probe rox qpcr master mix 2x
1
Quantification of Viral RNA in Human Fibroblasts
2
Quantitative RT-PCR for Viral RNA Quantification
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Total RNA was extracted from human fibroblasts by using Tri reagent (Sigma-Aldrich). The RNA pellet was resuspended in 25 μl of RNase-free distilled water and stored at -80 °C. The RNA was then used for reverse transcription using MMLV reverse transcription Kit (Promega) according to the manufacturer's instructions. The reaction was carried out using 1 μg total RNA as template for the normalization of viral RNA to the amount of total RNA. The MaximaTM Probe/ROX qPCR Master Mix (2x) (Thermo Scientific) was used in qPCR experiments. Each reaction of 25 μL contained 400 nM of each primer, 200 nM of specific probe and 1x Maxima Probe/ROX qPCR Master Mix.
Primers and probe sequences are listed in Table 1(Tab. 1) . The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real-time data were analyzed using the SDS software (Thermo Fisher Scientific). Viral RNA was quantified by comparing the sample's threshold cycle (Ct) values with each virus RNA standard curve which was obtained as previously described (Wichit et al., 2017[28 (link)]).
Primers and probe sequences are listed in Table 1
3
Quantification of Chikungunya Virus RNA
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Total RNA was extracted from SLC, MB and MT using Tri Reagent (Sigma-Aldrich). RNA was reverse transcripted using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Charbonnieres, France) according to the manufacturer's instructions. The reaction was carried out using 1 mg total RNA as template for the normalization of viral RNA to the amount of total RNA. The Maxima TM Probe/ROX qPCR Master Mix (2x) (Thermo Fisher Scientific, Illkirch, France) was used in qPCR experiment. Each reaction of 25 mL contained 400 nM of each primer, 200 nM of specific probe and 1x Maxima Probe/ROX qPCR Master Mix. The following CHIKV Primer Sequence (5'->30) were used:
CHIKV-P 50CCAATGTC (TC)TC (AC)GCCTGGACACCT3' [39] (link).
The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95°C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). Viral RNA was quantified by comparing the sample's threshold cycle (Ct) values with each virus RNA standard curve that was obtained as previously described [39] (link). Data are expressed as mean ± SEM of two independent experiments performed in triplicate.
CHIKV-P 50CCAATGTC (TC)TC (AC)GCCTGGACACCT3' [39] (link).
The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95°C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). Viral RNA was quantified by comparing the sample's threshold cycle (Ct) values with each virus RNA standard curve that was obtained as previously described [39] (link). Data are expressed as mean ± SEM of two independent experiments performed in triplicate.
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