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Protocol: cDNA Synthesis Using the RT2 First Strand
Kit
Use of the RT2 First Strand Kit is critical for obtaining optimal results and for
detection of the reverse transcription controls contained in the RT2 Predictor PCR
Array.
Important points before starting

Use the same amount of total RNA for reverse transcription of each
sample. First-time users are recommended to start with 300 ng total RNA
for each sample. Use of less than 100 ng RNA will result in a high rate of
false negatives that will affect the pathway activity regulation readout.

Do not use DEPC-treated water. Use high-quality, nuclease-free water.

The RT2 First Stand Kit is not compatible with the chemicals in DNA-free™
kits from Ambion. If your RNA sample has been treated with DNA-free
reagents, contact QIAGEN Technical Service.
Procedure
1. Thaw the reagents of the RT2 First Strand Kit. Briefly centrifuge (10–
15 s) to bring the contents to the bottom of the tubes.
2. Prepare the genomic DNA elimination mix for each RNA sample
according to Table 1. Mix gently by pipetting up and down and then
centrifuge briefly.
Table 1. Genomic DNA elimination mix
Component
RNA*
Buffer GE
RNase-free water
Total volume
Amount
100 ng–1 µg
2 µl
Variable
10 µl
* If using the kit for the first time, use the RNA amount recommended in “Important points
before starting” above.
3. Incubate the genomic DNA elimination mix for 5 min at 42°C, then
place immediately on ice for at least 1 min.
RT2 Predictor PCR Array Handbook 10/2012
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