Appendix - NEC express5800 A2010b User Manual

PrimeScript™ RT Master Mix （Perfect Real Time）
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Appendix

Preparation of RNA Samples
It is important to use a highly pure RNA samples for better cDNA yield. It is essential to
inhibit RNase activity in the cells and also to prevent RNase derived from equipment
and solutions used. Extra precautions should be taken during the sample preparation,
including use of clean disposable gloves, dedication of a exclusively table for exclusive
use for RNA preparation, and avoiding unnecessary speaking during assembly to prevent
RNase contamination from operator sweat or saliva.
A. Equipment
Disposable plastic equipment should be used. For glass tools should be treated
with the following procedure prior to use.
(1) Hot-air sterilization (180℃, 60 min.)
(2) Sterilize glassware in 0.1% diethyl pyrocarbonate (DEPC) solution at 37℃
It is recommended that all the equipment be used exclusively for RNA preparation.
B. Reagents
All reagents to be used in this experiment must be prepared using tools which
were treated as described in previous section (Hot-air sterilization (180℃, 60 min.)
or DEPC treatment), and all distilled water must be treated with 0.1％ DEPC and
autoclaved.
All reagents and distilled water should be used exclusively for RNA experiments.
C. Method of Preparation for RNA Samples
Use of highly purified RNA obtained by GTC (Guanidine thiocyanate) method, etc
is recommended. RNA isolation kits such as RNAiso Plus (Cat. #9108/9109) can also
be used for isolating high purity total RNA. The purified RNA sample should be
dissolved in sterilized distilled water or sterilized TE buffer.
D. Contamination with Genomic DNA and its countermeasure
In some cases, a total RNA sample may contain a small amount of genomic DNA,
which could potentially become a PCR template. This could result in inaccurate
results.
To avoid this to situation, either design primers which will not amplify genomic
DNA or remove genomic DNA by DNase I treatment.
(1) Designing a primer which will not amplify genomic DNA :
10
for 12 hours. Autoclave (120℃, 30 min.) to remove any residual DEPC.
It is possible to design a primer that will not amplify genomic origin
of DNA based on the exon and intron structure of genomic DNA. First,
confirm the target genomic structure, and select a large intron region.
Then design primers on the upstream and downstream sides of the
intron region. If the intron is large enough, genomic DNA amplification
will not occur. Even if size of the intron is not large, products resulting
from amplification of genomic templates would be larger than products
originating from cDNA, allowing identification by dissociation curve
analysis. However, this approach cannot be applied for single-exon
genes or genes that contain pseudogene. Moreover, species without
introns or new species without well-identified genomic structures could
demonstrate similar problems. In this case, we recommend performing
the DNase I treatment described in (2).
Cat. #RR036Q
v201204Da_2
URL:http://www.takara-bio.com