Safety information - Eurogentec

20
Guidelines for successful RT-qPCR
The RT reaction should be set up in a clean environment to avoid contamination; cleaning
solutions are available to avoid any RNase contamination. We also recommend working
with RNase free plastics. The tubes containing the reaction should be maintained on ice
during the set up of the reaction.
When setting up a qPCR reaction, it is always recommended preparing a Reaction mix
containing all the reagents required for the reaction in order to minimize the differences
across the 96-well plate and allow for more accurate pipetting (as the volume required
per sample is usually very small). The reaction mix should ideally be prepared in a
separate room from the one where DNA samples have been prepared in order to avoid
any contamination.
The DNA samples should always be added to the side of each well before the reaction mix
and then rinsed down while adding the reaction mix. As the reaction mix is heavier than
the DNA, the DNA will be mixed within the reaction mix. The plate can gently be shaken
on a plate shaker and spun before placing it on the machine. This is not an essential step
but will ensure that the reactions are thoroughly mixed and collected at the bottom of the
wells. It is useful to also check the wells for bubbles, as bubbles at the bottom of the well
can produce unusual plots on the results, when using machines that read from the top.
When sealing the PCR plates, it is important to ensure that it has been correctly done.
If optical films are used, fingerprints and marks should be avoided on the top of the film.
The qPCR machine used should be programmed according to the manufacturers
instruction manual.
The thermal cycling conditions described in this manual should be used as a good
starting point, but optimization of these conditions may be required for certain assays to
achieve optimal results.
Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I info@eurogentec.com