Quantitative RT-PCRs were used to validate RNA-Seq data. Reverse transcription PCR was achieved using a cDNA synthesis kit (Promega) according to the manufacturer's instructions. Specific RT-PCR primers were used to amplify central fragments of approximately 200 bp in length from different genes. For qRT-PCRs, quantification of gene expression and melting curve analysis were completed using a LightCycler (Roche) and Platinum SYBR Green qPCR Supermix-UGD (Invitrogen) was used according to manufacturer's instructions. The constitutively expressed housing keeping gene, 16S rRNA was used as a reference to standardize all samples and replicates.
Platinum sybr green qpcr supermix ugd
Manufactured by Thermo Fisher Scientific
The Platinum SYBR Green qPCR Supermix-UGD is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains the necessary components for efficient and sensitive DNA amplification using the SYBR Green detection method.
Automatically generated - may contain errors
3 protocols using platinum sybr green qpcr supermix ugd
1
Validating RNA-Seq Data with Quantitative RT-PCR
2
Validating RNA-Seq Data via Quantitative RT-PCR
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Quantitative RT‐PCRs were used to validate RNA‐Seq data. Reverse transcription PCR was achieved using a cDNA synthesis kit (Promega) according to the manufacturer's instructions. Specific RT‐PCR primers were used to amplify central fragments of approximately 200 bp in length from different genes. Semi‐quantitative RT‐PCRs were completed using 250 ng μl−1 cDNA template and PCR Mastermix (Promega) for 24–36 cycles. For qRT‐PCRs, quantification of gene expression and melting curve analysis were completed using a LightCycler (Roche) and Platinum SYBR Green qPCR Supermix‐UGD (Invitrogen) was used according to manufacturer's instructions. The constitutively expressed housing keeping gene, 16S rRNA was used as a reference to standardize all samples and replicates.
3
qPCR for Gene Expression Analysis
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qPCR reactions were set up in triplicates with each reaction having a total
volume of 10 μL containing 1X Platinum SYBR Green qPCR SuperMix-UGD
(Invitrogen), 0.2 μM forward and reverse primers and 1 μL cDNA. A CFX Connect™
Real-Time PCR Detection System (Bio-rad, USA) was used at standard cycling
parameters of 50°C for 2 min, 95°C for 2 min and then 95°C for 15 s and 60°C for
30 s for a total of 40 cycles. qPCR data were analysed using the delta delta
method (ΔΔCt) using GAPDH as the reference gene and expression in the Synthemax
II microcarriers as control.
volume of 10 μL containing 1X Platinum SYBR Green qPCR SuperMix-UGD
(Invitrogen), 0.2 μM forward and reverse primers and 1 μL cDNA. A CFX Connect™
Real-Time PCR Detection System (Bio-rad, USA) was used at standard cycling
parameters of 50°C for 2 min, 95°C for 2 min and then 95°C for 15 s and 60°C for
30 s for a total of 40 cycles. qPCR data were analysed using the delta delta
method (ΔΔCt) using GAPDH as the reference gene and expression in the Synthemax
II microcarriers as control.
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