Safety information - Eurogentec

8
Standard protocol
1. Reagent handling
• Thaw all required reagents completely and put them on ice.
• Mix all reagents well by inversion and spin them down prior to pipetting.
2. RNA preparation
The quality of the RNA is essential to the overall success of the all data from MiRmaid
miRNA precursors qRT-PCR primer set.
• Clean your work area before starting with RNAZap product or equivalent.
• Wear clean lab coat and gloves at any time (and change them whenever you suspect
that they are contaminated).
• Use RNase free tips and tubes.
Tips
The pre-miRNAs look highly sensitive to RNA degradation, therefore the quality of the
RNA should be checked before and after DNase treatment on an Agilent Bioanalyser
2100 if possible (or at least on agarose gel figure 2). A ratio 260/280 of 1.8 - 2.0 as well
as a perfect electropherogram are crucial to guarantee highly specific amplification.
Moreover, because of the small size of the pre-miRNAs, it is recommended not using
any total RNA extraction protocol based on exclusion column, as with this method all
small RNAs would be lost.
We recommend working on total
RNA extracted with Trizol or RNABle
RNA Extraction procedure. The
figure 3 shows the analysis of total
RNA after RNABle RNA Extraction,
data obtained with the Agilent
Bioanalyser 2100.
Figure 2: Results on agarose gel 2 %
TThhee ttoottaall RRNNAA iiss eexxttrraacctteedd wwiitthh tthhee RRNNAABBllee
pprroocceedduurree..
A smear around 150 bp shows the presence of
small RNAs after DNase treatment.
1 2 3 4
Before After
DNase treatment
Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I info@eurogentec.com
DNA ladder
4000 bp
800 bp
350 bp
150 bp