Procedure

1. Measure RNA via NanoDrop to determine volume of 1 ug.

2. cDNA Synthesis:

Prepare 16 ul RNA reaction [1 ug].
Add 4 ul of iScript RT supermix (BioRad 1708841) to 16 ul RNA reaction (total ~20 ul)

Ideally no-RT control supermix should be used to check for genomic DNA contamination

3. qPCR with SensiFast SYBR Kit

denature: 1: None
PCR cycles: 40: Quantification
melt: 1: Melting Curves
cool: 1: None
Target: 95; Acquisition Mode: None; Hold: 00:02:00; Ramp Rate: 4.8; Sec Target: 0; Step Size: 0; Step Delay: 0
Target: 95; Acquisition Mode: None; Hold: 00:00:05; Ramp Rate: 4.8; Sec Target: 0; Step Size: 0; Step Delay: 0
Target: 60; Acquisition Mode: Single; Hold: 00:00:20; Ramp Rate: 2.5; Sec Target: 0; Step Size: 0; Step Delay: 0
Target: 95; Acquisition Mode: None; Hold: 00:01:00; Ramp Rate: 4.8
Target: 40; Acquisition Mode: None; Hold: 00:01:00; Ramp Rate: 2.5
Target: 65; Acquisition Mode: None; Hold: 00:00:01; Ramp Rate: 1
Target: 95; Acquisition Mode: Continusous; Ramp Rate: 0.02; Aquisitions: 25
Target: 40; Acquisition Mode: None; Hold: 00:00:10; Ramp Rate: 2.5; Sec Target: 0; Step Size: 0; Step Delay: 0
create new experiment from template (SensiFast SYBR)
Save Experiment (floppy disk symbol)

Use 4titude white plate (BASIC Scientific 4ti-0381) on cooling plate on ice.
Dilute primer pairs to 5 uM per single primre (1:20, if stock 100 uM) in RNase-free water.

20 ul cDNA + 180 ul water = 1:10
dilution can be much higher (1:50); amount of cycles to reach c_p value is increased

4. Analyze qPCR with Lightcycler software

Recipes