Biomedical Engineering Reference
In-Depth Information
5.
RNase-Free DNase Set (Qiagen).
6.
RNase Away (Invitrogen).
2.3. Real-Time Quantitative RT-PCR
1.
Primers (
see
Note 1
).
a.
GAPDH
mRNA:
i. Forward primer: 5'-GAAGGTGAAGGTCGGAGT-3'.
ii. Reverse primer: 5'-GAAGATGGTGATGGGATTTC-3'.
b.
hPL
mRNA:
i. Forward primer: 5'-CATGACTCCCAGACCTCCTTC-3'.
ii. Reverse primer: 5'-TGCGGAGCAGCTCTAGATTG-3'.
2.
Dual-labeled fluorescent probes (
see
Note 1
):
a.
GAPDH
mRNA:
5'-(FAM)CAAGCTTCCCGTTCTCAGCC(TAMRA)-3'.
b.
hPL
mRNA:
5'-(FAM)TTCTGTTGCGTTTCCTCCATGTTGG(TAMRA)-3', where FAM
is 6-carboxy-fluorescein; TAMRA is 6-carboxy-tetramethylrhodamine.
3.
Calibrator:
a. TaqMan
®
Human
GAPDH
Control Reagents (Applied Biosystems, Foster
City, CA) (
see
Note 2
).
b. Synthetic DNA oligonucleotides specifying the
hPL
amplicon, HPLC-
purified (Proligos, Singapore) (
see
Note 3
):
5'-TGCGGAGCAGCTCTAGATTGGATTTCTGTTGCGTTTCCTCC
ATGTTGGAGGGTGTCGGAATAGAGTCTGAGAAGCAGAAGGAGGTCT
GGGAGTCATGC-3'.
4.
RNase-free water.
5.
EZ r
Tth
RNA PCR reagent set (Applied Biosystems).
6.
ABI Prism 7700 Sequence Detector (Applied Biosystems).
3. Methods
3.1. Sample Collection
1.
Collect blood samples in EDTA tubes. To ensure a sufficient amount of plasma
for RNA analysis, at least 4 mL of blood should be taken for each sample (
see
Note 4
). To guarantee a good quality of RNA, the blood samples should be
processed as soon as possible after venesection. If the processing procedures
cannot take place immediately, the blood samples should be stored with extra
care (
see
Note 5
).
2.
Centrifuge the blood samples at 1600
g
for 10 min at 4°C.
3.
Transfer plasma into new tubes.
4.
Perform the second round centrifugation at 16,000
g
for 10 min at 4°C (
see
Note 6
).
5.
Add 2 mL of Trizol LS reagent to 1.6 mL of plasma (
see
Note 7
) and vortex
vigorously.
6.
Store the Trizol-plasma mixture at -80°C until RNA extraction.
