Table 1. Reverse Transcriptase Reaction Volume Calculation Table (Section 3.1.5 Q-RT PCR, Step 2).
Insert number of desired samples and multiply by the modifiers indicated. Then multiply each reaction component by this number to calculate the total volume necessary to add to the RT reaction master mix. This should then be vortexed, divided evenly into the number of aliquots needed to measure the desired number of miRNAs. Primers should then be added per the protocol (Steps 3–5).
| Component | Volume/sample (μl) | X | Number of Samples x number of miRNA to be measured + 1(control) x 1.2 | = | Total |
|---|---|---|---|---|---|
| 10× RT Buffer | 1.36 | x | = | ||
| dNTP Mix (100 mM) | 0.14 | x | = | ||
| MultiScribe™Reverse | 0.91 | x | = | ||
| Transcriptase | |||||
| RNase Inhibitor | 0.17 | x | = | ||
| Nuclease-free H2O | 7.42 | x | = |