Hepatitis B Virus (HBV)

Quantitative Gonadotropin-Releasing Hormone: MATERIAL AND METHODS(9)

PCR for /3-Actin

Five-microliter aliquots from each RT product were mixed with 95 ^l of the PCR-mastermix described above with 4 ^l 3′ + 5′ primer-mix (5 ^M each) for p-actin. Program parameters were 30 cycles of 94°C for 45 sec, 54°C for 45 sec, and 72°C for 60 sec. For this PCR, no competitor was added. flovent inhaler

Nested PCR for the GnRH Receptor

RT products were split for the first round of PCR. Two microliters of RT product was added to 48 ^l of PCR-1-mix (2 mM MgCl2 solution, 10-strength PCR buffer II, 33.5 ixl DEPC-treated dH2O, 0.24 mM of each dNTP, 2.5 U polymerase gold [all Perkin-Elmer], and 2.4 ^l outer 3′ + 5′ primer-mix [5 ^M each, Table 1]). After all components were mixed in a 0.5-ml thin-wall PCR cup, the reaction mix was covered with 50 ^l light white mineral oil. After completion of 30 cycles of 94°C for 45 sec, 54°C for 45 sec, and 72°C for 60 sec, the reaction was terminated at 72°C for 5 min and cooled down to 4°C. First-round PCR products were stored at -20°C until the second PCR. For the second PCR, 5 ^l of first-round PCR products was added to 95 ^l PCR2 reaction mix (1.9 mM MgCl2 solution, 10-strength PCR buffer II, 65.0 ^l DEPC-treated H2O [dist.], 0.2 mM of each dNTP, 2.5 U polymerase gold [all Perkin-Elmer], and 4 ^l inner 3′ + 5′ primer mix [5 each, Table 1]), in a thin-wall PCR tube, and covered with 50 ^l mineral oil. Program parameters were identical with those of the first round of PCR. After the second round of PCR was completed, samples were stored at -20°C until agarose gel electrophoresis was carried out.

Data Analysis

Statistical analysis was performed by ANOVA and independent sample £-test. The analysis was carried out using the Statistical Package for Social Science (SPSS Inc., Chicago, IL) with a p value < 0.05 considered statistically significant.
TABLE 1.

Size of Position of 3’/-
amplified primers on 5′-
cDNA Ref # fragment cDNA end* Sequence of oligonucleotide
p-Actin 48 838 bp 294-325 5′ 5′ ® ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG ® 3′
1100-1131 3′ 5′ ® CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC ® 3′
GnRH 49 399 bp 47-67 5′ 5′ ® AAA ACT CCT AGC TGG CCT TAT ® 3′
445-426 3′ 5′ ® GGA ATA TGT GCA ACT TGG TG ® 3′
445-426 3’C 5′ ® GGA ATA TGT GCA ACT TGG TGA GCG TTG GGT TTC TGC ® 3′
+213-198
GnRH-receptor 50 524 bp 300-319 5’o 5′ ® GAT TGT CAT GCC ACT GGA TG ® 3′
823-801 3’o 5′ ® TTA GAG TCT TCA GCC GTG CTC ® 3′
231 bp 477-496 5’i 5′ ® CAA CAG CAA AGT CGG ACA GT ® 3′
706-687 3’i 5′ ® AGC ATG AAA AGA GGG ATG ATG ® 3′

Category: Hormone Gene Expression

Tags: Gene Expression, Gonadotropin, Hormone, Human Endometrium, Menstrual Cycle

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