Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 13)

| 7 SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual | 7
4. Dilute the cDNA as needed to perform real-time PCR reactions using a minimum of two
genes — reference and low expressing. However, it is recommended to evaluate four genes
— reference, low, medium, and high expressing.
5. Prepare the real-time PCR plate (Figure 3) and cycle according to the recommended protocol.
Fig. 2. Tenfold serial dilution of RNA starting at 1 µg down to 1 pg,
thus covering six logs of dynamic range. Each RNA dilution was
transferred to the respective cDNA reaction tube for cDNA synthesis.
Fig. 3. A recommended plate layout.
Reference
gene
Medium
expressor
Low
expressor
High
expressor
10 0
1 µg RNA
Reaction 1
100 ng RNA
Reaction 2
10 ng RNA
Reaction 3
1 ng RNA
Reaction 4
100 pg RNA
Reaction 5
10 pg RNA
Reaction 6
1 pg RNA
Reaction 7
10 –1
10 –2
10 –3
10 –4
10 –5
10 –6
Serial dilution
of the RNA
Bio-Rad ® iScript ™ cDNA
Synthesis Kit