Mj mini thermal cycler

Quantitative real-time PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad) and run in triplicate in 48-well plates with an MJ Mini Thermal Cycler (Bio-Rad). The cycling profile consisted of 95°C for 1 min, followed by 40 cycles of 5 s at 95°C and 20 s at 60°C, as recommended by the manufacturer. The amplification process was immediately followed by a melting curve analysis steps of 0.5°C every 10 s from 65°C to 95°C. Baseline and threshold cycles (Ct) were automatically determined using the Bio-Rad CFX manager software (Bio-Rad). Here, we selected a classical housekeeping gene, Actin, and a novel housekeeping gene, CAC [37] (link) (Table1) as internal controls for constitutive expression in various tissues.

Total RNA was isolated from manually dissected ovaries using Trizol reagent (Invitrogen, Thermo Fisher Scientific) and cleared of genomic DNA by DNA-free kit (Ambion).
For analysis of signaling pathway genes in germaria, RNA was isolated from 0-1-day ovaries containing no late stage egg chambers. 1 μg of total RNA was used for the reverse transcription reaction with oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen). The resulting cDNAs in at least three biological replicates were analyzed by RT-qPCR performed in MJ Mini thermal cycler (Bio-Rad) using SYBR Green chemistry (Applied Biosystems). The following primers were used for PCR:
Gypsy for CTTCACGTTCTGCGAGCGGTCT,
Gypsy rev CGCTCGAAGGTTACCAGGTAGGTTC,
Zam for3 TCACATCCTTCCAGCAATCTTCAA,
Zam rev3 TATTACAGTTTCTGACATTATTTCTTCGTG,
MDG1 dir AACAGAAACGCCAGCAACAGC,
MDG1 rev CGTTCCCATGTCCGTTGTGAT,
Idefix for AACAAAATCGTGGCAGGAAG,
Idefix rev TCCATTTTTCGCGTTTACTG,
dpp for2 GGCTTCTACTCCTCGCAGTG,
dpp rev2 TGCTTTTGCTAATGCTGTGC,
wnt4 for5 ATGATCCTCACCCACCTGAG,
wnt4 rev5 ACCTGACCAGCATTGTTTCC,
wnt2 for CAATAACCGAGCAGGGAGAAC,
wnt2 rev CATGAGTCTATCGCCAACCAG,
fz3 for TCTGCTTCGTCCTGACACTG,
fz3 rev CCTTGCTTGATTGTGGAACAC,
Rp49_up ATGACCATCCGCCCAGCATAC,
Rp49_rev2 GCTTAGCATATCGATCCGACTGG.

In the next step of the analysis, a qualitative assessment of
ACVR2A gene expression was performed. In this analysis an
ACVR2A gene fragment was amplified by polymerase chain
reaction. The PCR reaction was carried out using ready-made solution containing
Taq DNA polymerase, dNTPs, MgCl₂ and reaction
buffers (Biotool, Germany), according to the manufacturer’s protocol. The 20 μL
reaction mixture for PCR consisted of 5 μL of 2x PCR Super Master Mix (Biotool,
Germany), 0.7 μL of each primer at a concentration of 0.5 μM (sequences for the
ACVR2A primer set were: forward
5’-AGGGTTCACTATCAGACTTTC-3’; reverse 5’-GTAAATATGCCAATCCTCTAGC-3’), 1 μL of cDNA
template, and distilled water to a final reaction volume of 20 μL. In every
experiment, a negative control sample (reaction mixture without cDNA template)
was used. The PCR reaction conditions included the initial denaturation for 5
min at 95 °C, 34 cycles consisting of 3 steps (denaturation for 1 min at 95 °C,
annealing for 1 min at 57 °C and elongation for 1 min at 72 °C) and final
elongation for 7 min at 72 °C. PCR amplifications were carried out in MJ Mini
Thermal Cycler (Bio-Rad, USA). The presence of 96 bp PCR products for the
ACVR2A gene was assessed by electrophoresis in 2% agarose
gels. For quantitative assessment, only samples that showed the mRNA expression
of ACVR2A gene in qualitative analysis were included.