Biology Reference
In-Depth Information
3.8.4. Perform Quantitative
PCR Reactions
7. Real-time quantitative PCR is performed with SYBR green as
a double-strand DNA-specifi c dye in Eppendorf Mastercycler
EP Realplex (Eppendorf North America Inc., Westbury, NY,
USA, Fig.
2b
).
8. The primers for rat hemoglobin a-chain (HbA) and hemoglobin
b-chain (HbB) are designed from known sequences of rat HbA
mRNA (GenBank no. U29528) and HbB mRNA (GenBank
No. NM_033234) searched by PrimerQuest (Integrated DNA
Technologies Inc., Coralville, IA, USA). Rat HbA oligonucle-
otide primer sequences are 5¢-TGA TCC ACT TCC TTC TCT
GCC CAA-3¢ (forward primer) and 5¢-ATC AGT TGC CCA
AGT GCT TCT TGC-3¢ (reverse primer). The primer
sequences for HbB are 5¢-ATG GCC TGA AAC ACT TGG
ACA ACC-3¢ (forward primer) and 5¢-TGG TGG CCC AAC
ACA ATC ACA ATC-3¢ (reverse primer). Rat GAPDH, a
“housekeeping gene,” serves as a control. Its primers are 5¢-
CCG TGC CAA GAT GAA ATT GGC TGT-3¢ (forward) and
5¢-TGT GCA TAT GTG CGT GTG TGT GTG-3¢ (reverse).
9. PCR is run in triplicate on 96-well plate with a total volume of
20 mL per well using 2.5× SYBR
®
Green universal master mix.
10. Cycling conditions are 2 min at 95°C, 30 s at 95°C, 30 s at
60°C, 1 min at 72°C, 40 cycles and a melting curve program
(60°C to 95°C with warming of 1.75°C/min).
11. The relative quantifi cation analysis module is used to compare
expression levels of the target gene.
3.9. Sequencing
of the PCR Products
PCR products are isolated by 2% agarose gel electrophoresis. Gels
are observed with ethidium bromide staining and ultraviolet tran-
sillumination. The DNA fragments of interest are excised and then
purifi ed with an Agarose Gel DNA Extraction kit (Roche,
Diagnostics GmbH, Mannheim, Germany). Purifi ed DNA frag-
ments are sequenced. We perform sequencing on an Applied
Biosystems DNA Sequencer (Model 3730XL, Foster City, CA,
USA). Sequence data are analyzed using the nucleotide blast at
National Center for Biotechnology Information.
References
1. Towbin H, Staehelin T, Gordon J (1979)
Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets:
procedure and some applications. Proc Natl
Acad Sci U S A 76(9):4350-4354
2. Burnette WN (1981) “Western blotting”:
electrophoretic transfer of proteins from
sodium dodecyl sulfate-polyacrylamide gels
to unmodifi ed nitrocellulose and radiographic
detection with antibody and radioiodinated
protein
A.
Anal
Biochem
112(2):
195-203
3. Wu J, Hua Y, Keep R, Schallert T, Hoff J, Xi G
(2002) Oxidative brain injury from extravasated
erythrocytes after intracerebral hemorrhage.
Brain Res 953(1-2):45
4. Wu J, Hua Y, Keep RF, Nakamura T, Hoff JT,
Xi G (2003) Iron and iron-handling proteins in
the brain after intracerebral hemorrhage. Stroke
34(12):2964-2969
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