Biomedical Engineering Reference
In-Depth Information
3.2. RNA Extraction
The RNA extraction should be performed in a clean area to minimize RNase
contamination. RNase Away can be used to clean lab bench and equipment
before each extraction.
In this protocol, total plasma RNA is extracted.
1.
Thaw the Trizol-plasma mixture immediately before extraction.
2.
Add 0.4 mL of chloroform to the mixture (2 mL Trizol LS reagent and 1.6 mL
plasma) and mix vigorously.
3.
Centrifuge the mixture at 12,000
g
for 15 min at 4°C.
4.
Transfer the upper aqueous layer into new tubes. Add one volume of 70% etha-
nol to one volume of the aqueous layer.
5.
Apply the mixture to an RNeasy Mini column and wash the column according to
the manufacturer's recommendations.
6.
Perform the “On-Column DNase Digestion” with the RNase-Free DNase Set
according to the manufacturer's recommendations (
see
Note 8
).
7.
To elute RNA, add 30
L of RNase-free water into the RNeasy column and incu-
bate for 5 min at room temperature. Centrifuge the column for 1 min at 16,000
g
.
µ
8.
Repeat the elution step (
step 7
) by using the first eluate.
9.
Store the extracted RNA at -80°C.
3.3. Real-Time Quantitative RT-PCR
Plasma RNA is quantified by using one-step real-time quantitative RT-PCR
(
13
)
. In this method, the r
Tth
(
Thermus thermophilus
) DNA polymerase func-
tions both as a reverse transcriptase and a DNA polymerase
(
19
)
(
see
Note 9
).
In this protocol, the quantification of
GAPDH
(
11
)
and
hPL
(
17
)
mRNA is
described as follows.
3.3.1. Quantification of GAPDH mRNA
1.
Prepare a calibration curve for
GAPDH
RT-PCR system with the use of the human
control RNA supplied in the TaqMan Human
GAPDH
Control Reagents. The con-
trol RNA is serially diluted into concentrations ranging from 15 ng to
1.5 pg (
see
Note 2
).
2.
Set up the RT-PCR reaction mixture for the
GAPDH
mRNA according to
Table 1
.
3.
Add 3
L of sample RNA, control RNA (for calibration curve) or RNase-free
water (for negative blanks) into the reaction mixture.
µ
4.
Perform the real-time
GAPDH
RT-PCR reactions in the ABI Prism 7700 Sequence
Detector with cycling conditions show in
Table 2
.
3.3.2. Quantification of hPL mRNA
1.
Prepare a calibration curve by serially diluting the synthetic DNA oligonucle-
otides specifying the
hPL
amplicon with concentrations ranging from 2.5
×
10
7
copies/
µ
L to 25 copies/
µ
L (
see
Note 3
).
