Abi 7500 real time pcr detection system

The expression level of genes was detected using SYBR Premix Ex Taq II (Takara, China), and GAPDH was used as an internal control. qRT-PCR was performed on an ABI7500 real-time PCR detection system (Applied Biosystems). The signal was normalized to the internal control and expressed as 2^-ΔΔct. The primer sequences were listed in Table S2.

Total RNA was isolated from fetal hippocampus using TRIzol^® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The first strand of cDNA was produced using a Primescript™ RT reagent kit with genomic DNA Eraser (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. mRNA was quantified by qPCR using SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.) and a ABI 7500 Real-Time PCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was performed according to a previously described method (30 (link)). Using 20 µl of the reaction system, the reaction conditions were: Pre-denaturing at 95°C for 30 sec, and 45 cycles of 95°C for 5 sec and 63–68°C for 30 sec (Table I). Primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA), and their sequences are presented in Table I. The mRNA level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured and used as a quantitative control. The 2^−∆∆Cq method was used to quantify the expression of target genes (31 (link)). Experiments were repeated twice.

Total RNA from cell lines was extracted by TRIzol (Invitrogen; Thermo
FisherScientifc, Inc.) reagent according to the manufacturer’s protocol. After
obtaining the complementary DNA (cDNA) through reverse transcription using
PrimeScript RT (TaKaRa) in accordance with manufacturer’s manual, the mRNA
expression level of MYO10 was determined by quantitative real-time polymerase
chain reaction (qRT-PCR) using SYBR Premix Ex Taq kit (TaKaRa) on an ABI-7500
Real-Time PCR Detection System (Applied Biosystems) following amplification
conditions: Predegeneration at 95 °C for 5 minutes, followed by 40 cycles at 95
°C for 30 seconds, 45 seconds at 60 °C, and 30 minutes at 72 °C for the final
extension. GAPDH was used as the internal control, and the primer sequences were
as follows: MYO10: forward, 5′-TTCATGGACTTGCTCATCAGG-3′, and reverse,
5′-GTCCTCAGCTGTGTGTGACTT-3′; GAPDH: forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′, and
reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. All reactions were performed in
triplicate, and relative expressions of MYO10 in cell lines were calculated by
the 2^−ΔΔct method.

Total RNA from the tissues and cell lines were extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A total of 2 µg RNA was reverse-transcribed into cDNA using a PrimeScript First Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. qPCR was performed on an ABI 7500 Real-time PCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction conditions were 95°C for 10 min, and 35 cycles of 95°C for 30 sec and 60°C for 1 min. GADPH was used as the internal reference gene. To analyze the levels of miRNA expression, qPCR was performed using an All-in-One™ miRNA qRT-PCR kit (GeneCopoeia, Inc., Rockville, MD, USA), according to the manufacturer's protocol. The U6 gene was used as an internal control. The relative mRNA level was calculated using the 2^−∆∆Cq method (17 (link)). The primers used were as follows: Let7b forward, 5′-TGAGGTAGTAGGTTGTGTGGTT-3′; and reverse, 5′-GCTGTCAACGATACGCTACCTA-3′; GAPDH forward, 5′-CCGTCTAGAAAAACCTGCC-3′; and reverse, 5′-GCCAAATTCGTTGTCATACC-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′; and reverse 5′-AACGCTTCACGAATTTGCGT-3′ (all GeneCopoeia, Inc.).