Biology Reference
In-Depth Information
The relative expression of the target gene is compared to the
control. The difference in the expression is calculated as follows:
Δ
Ct = Ct target gene − Ct HKG.
Ratio = 2 −
Δ
Ct.
This calculation method assumes that the amount of DNA in
each cycle doubles; therefore, the real-time PCR effi ciency is
optimal.
4
Notes
1. For quantitative mRNA expression analysis, the tissue speci-
mens should be fi xed until processed in RNAlater
®
Stabilization
Reagent. This step is necessary to prevent the denaturation of
the mRNA by ribonucleases (RNases). RNases are stable
enzymes that exhibit high endonuclease activity that specifi -
cally cleaves RNAs. These enzymes are produced by all living
organisms and are found in practically every location.
2. After harvesting, the tissue specimen should be transferred
immediately to RNAlater stabilization reagent for storage. The
ratio of tissue to fi xative solution should be at least 1:6,
although a 1:10 ratio is ideal.
3. Gloves should be worn at all times when handling RNAs to
prevent the contamination of the specimen with RNases.
4. Without exception, consumables (pipette tips, Eppendorf
tubes, etc.) should be RNase free.
5. Prior to the chemical treatment, the fi xed tissue should fi rst be
crushed mechanically.
6. For the precipitation of nucleic acids, isopropanol is more effi -
cient than 70 % ethanol.
7. For the second round of DNase I treatments, we recommend
using a different manufacturer because enzymes from different
companies often differ greatly in their quality and effects.
8. For successful amplifi cation, the primers should meet the fol-
lowing criteria:
●
Primer length: 16-30 bp; 20-25 bp is optimal.
●
GC content: 50-60 %.
●
Primer Tm: 58-60 °C; the maximum Tm difference
between the two primers should not exceed 2 °C.
9. The primers should be tested for mRNA specifi city using the
BLAST method (
http://blast.ncbi.nlm.nih.gov/Blast.cgi
) to
prevent nonspecifi c primer binding and therefore nonspecifi c
gene amplifi cation.
