Safety information - Eurogentec

12
40 Cycles 15 sec 95 °C
1 min 60 °C
Data collection should be done at 60°C.
Perform a meltcuve to ensure the specificity of the product amplified.
Dissociation Curve 15 sec 95 °C
15 sec 60 °C
(Ramping time: 19’59’’ with fluorescence reading)
15 sec 95 °C
5. How to set up a good assay
RNA quality
The quality of the RNA is essential to the overall success of the all data from MiRmaid
miRNA precursors qRT-PCR primer set. Clean laboratory practice is important to
avoid degradation of the RNA (please find more information in paragraph 2 of the
standard protocol). As the pre-miRNAs look highly sensitive to RNA degradation it is
very important to check the quality of RNA before and after DNase treatment on an
Agilent Bioanalyser 2100 if possible or at least on an agarose gel (please find more
information p.8). Also, it is important to ensure that small RNAs have been well and
correctly extracted using the 5S and U6 positive controls (please find more
information concerning positive controls below).
No-Reverse Transcriptase control
As indicated in step 2 of the DNase and Reverse Tanscription Reactions paragraph, it
is recommended performing a No-Reverse Transcriptase control.
This control assumes the total digestion of the genomic DNA, which could interfere
with the cDNA amplification. This control includes all of the qPCR reagents with
primer controls and the RNA treated by DNase without the reverse transcription step.
If a product is amplified, it indicates that there is still some genomic DNA
contamination.
Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I info@eurogentec.com