REF 1N005 STAT-NAT® RNA-Mix EN

For further information regarding the thermal profile PRECAUTIONS IN USE

configuration, refer to the technical manual of the instrument In addition to the possible risks regarding the reactive components,
in use. product may contain non-reactive components such as
Segment Cycle Number Temperature Time preservatives (i.e. sodium azide or other) and detergents. The total
concentration of these components is lower than the limits reported
1 1 48 °C 30 min by the 67/548/EEC, 1999/45/EC directives and EC 1272/2008
2 1 95 °C 10 min Regulation (CLP) and related modifications regarding classification,
95 °C 30 sec labelling and packaging of dangerous preparations have been
made accordingly. However, it is recommended that reagents be
3 30-45 Tm – 5 °C 30 sec handled carefully, that ingestion and contact with eyes, skin and
mucous membranes be avoided and that laboratory reagents are
72 °C 30 sec used according to good laboratory practice.
4 1 72 °C 3-5 min Manage and waste all the biological samples as potentially
infectious. All the material that come in contact with the biological
Table B sample must be treated with 0.5% sodium hypochlorite for at least
9. perform real-time PCR analysis according to the manual of 30 minutes or sterilized in autoclave at 121°C for 30 minutes and
the software in use. The products obtained with the kit can be then wasted. Always use personal protective equipment for the
successfully used in post-amplification analysis, such as individual protection. Read all the instructions contained in the kit
Melting Curve Analysis, High Resolution Melting. insert before performing the test. Strictly attain to the insert kit
indication. Do not eat, smoke, or drink in the working area. Comply
 Real-time PCR Method with probe(s) (possible with the kit shelf life.
applications: gene expression analysis, viral/bacterial
detection): WASTE MANAGEMENT
1. Extract RNA from the samples to be examined (extraction Reagents must be disposed in accordance with local regulations.
system not included in the kit); BIBLIOGRAPHY
2. prepare the positive and negative controls (not provided); 1) Bustin SA. Absolute quantification of mRNA using real-time
3. before starting the reaction, turn on the equipment (Real-Time reverse transcription polymerase chain reaction assays. J Mol
PCR thermalcycler and computer) and open the dedicated Endocrinol. 2000 Oct;25(2):169-93.
software program; 2) Bustin SA. Quantification of mRNA using real-time reverse
4. set the detectors choosing the reporter(s) and quencher(s) transcription PCR (RT-PCR): trends and problems. J Mol
dye; this kit does not contain a passive reference dye; Endocrinol. 2002 Aug;29(1):23-39.
5. remove the necessary number of test tubes from the kit; 3) Bustin SA, Mueller R. Real-time reverse transcription PCR (qRT-
6. add the following components to the lyophilized mix present in PCR) and its potential use in clinical diagnosis. Clin Sci (Lond).
the tube: 2005 Oct;109(4):365-79.
- forward and reverse primers; 4) Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a
- labeled probe(s); polymerase-catalyzed chain reaction Methods Enzymol. 1987;
- RNA template; 155:335-50.
- PCR-grade RNase free water. 5) Wong ML, Medrano JF. Real-time PCR for mRNA quantitation.
The final reaction volume must be 25 µL. Biotechniques. 2005 Jul;39(1):75-85.
The lyophilized mix will dissolve in few seconds. 6) Patent No. WO2010133628 A1. A dried and stabilized ready-to-
7. make sure that there are no air bubbles; if so, remove them use composition containing nucleic acid polymerization enzymes
by aspiration with the tip of the pipette; for molecular biology applications.
8. perform real-time PCR using the thermal profile optimized for
primers and probe(s). The parameters suggested for the use
of STAT-NAT® RNA-Mix with Applied Biosystems thermal
cyclers and equipments are an example and are summarized
in Table C. The segment #3 of the cycling profile can be
arranged on the basis of the probe(s) used. For further
information regarding the thermal profile configuration,
refer to the technical manual of the instrument in use.
Segment Cycle number Temperature Time
1 1 48 °C 30 min
2 1 95 °C 10 min
95 °C 30 sec
3 30-45 Tm – 5 °C 30 sec

72 °C 30 sec
Table C
9. perform real-time PCR analysis according to the manual of
the software in use.
The examples reported here are for a non-comprehensive
and illustrative purpose only. STAT-NAT® mixes could also
be used in other applications, in addition to the here-
described ones; the use depends on the needs of the
laboratory technician. STAT-NAT ® is a trademark in various jurisdictions which is exclusively licensed to
SENTINEL CH. SpA. STAT-NAT® technology is covered by patent No.
QUALITY CONTROL WO2010133628 A1.
Use control materials with different concentration levels in order Note: changes in comparison to the previous version are indicated by a vertical
to verify assay accuracy. Quality control must be prepared by bar in the text margin.
operator using, where possible, approved and standard controls.

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