Bio-Rad SsoAdvanced™ Universal Probes Supermix - Manual (Page 15)

| 9 SsoAdvanced ™ Universal Probes Supermix Instruction Manual | 9
Determining the Dynamic Range of the Reverse Transcription Reaction
An optimal reverse transcription reaction is expected to generate a true representation of the
RNA converted into cDNA. However, it is imperative to determine the dynamic range of the
reaction to ensure that the initial RNA loaded does not fall outside the dynamic range. If it
does, then the downstream real-time PCR data may be invalid. To validate the dynamic range,
perform the following :
1. Preparation of a serial dilution using a single RNA source (or a pooled RNA sample) is
required to prepare the cDNA synthesis reactions for the experiment. Ensure an adequate
amount of RNA is available; adjust concentrations and volumes accordingly.
2. Start with 1 µg of total RNA and perform a tenfold serial dilution covering at least 5 or 6 logs
of dynamic range.
3. Perform RT using 20 μl reactions. Transfer the RNA, as shown in Figure 4, to the respective
reaction tubes. For example, transfer 1 μg of RNA to Reaction 1 tube. Repeat transferring
RNA to the remaining reaction tubes.
Fig. 4. 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.
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