iScript™ Reverse Transcription Supermix for RT-qPCR

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Prepare transfection master mix and mix throughly by pippetting. Add 15ul master mix to 5ul of RNA for each RT reaction. Adjust the volume of water if the input of RNA is not to 5 ul input (1ug-1pg). Transcribe for 30 min at 42 °C and the reaction is inactivated for 5 min at 85 °C.

Protocol tips
Prepare transfection master mix and mix throughly by pippetting. Add 15ul master mix to 5ul of RNA for each RT reaction. Adjust the volume of water if the input of RNA is not to 5 ul input (1ug-1pg). Transcribe for 30 min at 42 °C and the reaction is inactivated for 5 min at 85 °C.

Publication protocol

For a two-step RT-qPCR, RNA was extracted at corresponding time points using PureLink RNA Mini Kit (Thermo Fisher Scientific). The total RNA amount was quantified using Nanodrop (Thermo Fisher Scientific). 1 µg RNA was firstly primed for 5 min at 25 °C, then reverse-transcribed using iScript™ Reverse Transcription Supermix (Bio-Rad, US) for 30 min at 42 °C and the reverse-transcriptase was inactivated for 5 min at 85 °C. The subsequent cDNA was diluted 1:10 in nuclease-free water and stored at −20 °C. For the qPCR, reaction mixtures (10 µl) contained 5 µl of SsoFast™ EvaGreen® Supermix (Bio-Rad), 0.4 µM of the respective forward and reverse primers, and 2 µl of the thawn cDNA. After an initial denaturation cycle (98 °C for 2 min) the product was amplified in 40 PCR cycles (98 °C for 2 s, 60 °C for 5 s) followed by a melting curve analysis using the Roche LightCycler® 96 System (Roche, Switzerland).

For a ‘zero-step’ RT-qPCR, cells were lyzed directly in the culture well using VolcanoCell2G Lysis Buffer (myPOLS Biotec) for 15 min at 4 °C. Cell lysate dilutions were prepared in the lysis buffer and stored at −80 °C. Thawed diluted supernatant from approximately 1500 cells was then used as a template for the RT-qPCR reaction. Reaction mixtures (10 µl) contained 5 µl of VolcanoCell2G 2× RT-PCR Master Mix (myPOLS Biotec), 0.1 µM of the respective hydrolysis probe, and 0.4 µM of the respective forward and reverse primers. The amount of template in each reaction was equivalent to the amount of mRNA in approximately 1500 cells. After an initial denaturation step (95 °C for 3 min) the product was amplified in 40 PCR cycles (95 °C for 15 s, 62 °C for 75 s). Real-time quantification was performed using hydrolysis probes (TaqMan™ probes). A detailed protocol for the ‘zero-step’ RT-qPCR is provided in the Supplementary Material.

In general, all qPCR reactions for each target gene were set up manually in triplicates of three biological replicates (three independent cell differentiations) from day 0 to day 8 LUHMES. Primers, probes, and their targets are described in the Supplementary Material.

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Manufacturer protocol

Download the product protocol from Bio-Rad Laboratories for iScript™ Reverse Transcription Supermix for RT-qPCR below.

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