Rotor gene 6000 system

As angiogenesis-related genes were expected to be
expressed following successful transplantation of ovarian
tissue in the mice, VEGF and Ang-2 primers were
designed for evaluation of the genes. For this purpose,
all fresh and vitrified/grafted ovaries were immediately frozen in liquid nitrogen and stored at -196˚C for real-time
polymerase chain reaction (PCR) analysis. The ovaries
were collected for RNA extraction by Trizol Reagent
(Invitrogen, USA) according to the manufacturer’s
recommendations. The specimens were treated with
RNase-free DNase and single-stranded cDNAs and were
synthesized by incubating 1 μg of isolated RNA. The realtime
PCR analysis was carried out by the Corbett Life
Science (Rotor-Gene 6000) System and Fast Start SYBR
Green Master (Roche). Primer sequences for VEGF and
Ang-2 are outlined in Table 1.
Real-time PCR amplifications were performed using the following program: denaturation
of cDNA (1 cycle at 95˚C for 10 minutes), amplification (40 cycles at 95˚C for 15
seconds, 57˚C for 30 seconds and 63˚C for 38 seconds), and melting curve analysis (1
cycle at 60 to 95˚C with 1˚C/seconds). The mRNA expression levels were normalized by
β-microglobulin (β-mg) and the quantification was evaluated using the
2^(∆∆Ct) method. The assay was performed in triplicate.

The expression of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was measured using a real-time PCR technique (Table 1). The samples obtained from different groups were immediately frozen in liquid nitrogen and stored at -196˚C. RNA extraction was done by Trizol Reagent (Cat no: 302-001; Gene All) and reverse-transcribed to cDNA using an RNase-free DNase synthetase kit (Cat no: PP-410 S; Jena Bioscience). The PCR reaction was done using Corbett Life Science (Rotor-Gene 6000) System and Fast Start SYBR Green Master (Roche). Real-time PCR amplifications were performed using the following program: denaturation of cDNA (1 cycle at 94˚C for 10 min), amplification (40 cycles at 95˚C for 10 s, 60˚C for 35 s, and 72˚C for 20 s), and melting curve analysis (1 cycle at 60 to 95˚C with 1˚C/seconds). The 2^−ΔΔCT method was used to evaluate the quantity.
Table 1

Primers list

Gene	Forward	Reverse	Annealing (°C)
ang-1	5′-GCCACTTGAGAATTACATTGTGG-3′	5′-CGCGGATTTTATGCTCTAATCAACTG-3′	59
ang-2	5′-GTCTCCCAGCTGACCAGTGGG-3′	5′-TACCACTTGATACCGTTGAAC-3′	59
gapdh	5′-CTCTAAGGCTGTGGGCAAGGTCAT-3′	5′-GAGATCCACCACCCTGTTGCTGTA-3′	59