Abi 7500 real time pcr

Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For quantitative analysis of miR-Let7A, reverse transcription (RT) was performed using Taqman Micro RNA RT kit (Takara, Otsu, Shiga, Japan) according to the manufacturer's instructions with 10 ng total RNA. PCR reactions were then performed according to manufacturer's instructions to quantify the expression level of miR-Let7A using Taqman Universal PCR Master Mix, No Amp Erase UNG (Applied Biosystems, Foster City, CA, USA), and Taqman microRNA assay (Takara, Otsu, Shiga, Japan) for the miR-Let7A of interest. The PCR amplification was performed in ABI 7500 Real Time PCR (Life Technologies Corporation, CA, USA) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The PCR incubation profile was extended to 40 cycles for miR-Let7A. The PCR primers for Let-7A were (F): 5′-GCGCCTGAGGTAGTAGGTTG-3′ and (R): 5′-CAGTGCAGGGTCCGAGGT-3′. The PCR primers for U6 were (F): 5′-CTCGCTTCGGCAGCACATATACT-3′ and (R): 5′-ACGCTTCACGAATTTGCGTGTC-3′, respectively. The data were uniformly normalized to the internal control U6, and relative expression levels were evaluated using the 2^−ΔΔCt method. All experiments were run in triplicate (Zhang et al., 2014 (link)).

After the mice were sacrificed, bilateral hippocampi were quickly separated and placed in a glass homogenizer. Total mRNA was extracted according to Trizol reagent instructions. cDNA synthesis was performed by RT-PCR reverse transcription kit (MedChemExpress, USA) operation. PCR amplification mixed system (Lifeint, Xiamen, China) was conducted on the ABI7500 Real-Time PCR (Thermo Fisher Scientific, USA). The internal reference gene was GAPDH and the gene level was determined utilizing the 2^−ΔΔCt method. The primer sequences were listed in Table 1.

Total RNA was isolated from cells using QIAshredder and RNeasy Mini Kit (Qiagen, Hombrechtikon, Switaerland). cDNA synthesis and amplification was performed using the Superscript III first-strand synthesis kit according to manufacturer's instructions (ThermoFisher). Quantitative real-time PCR (qPCR) analysis was performed with the Fast SYBR Green Master Mix (ThermoFisher) and the ABI7500 real-time PCR (ThermoFisher). Data was normalized to 18S rRNA. See Supplemental Table I for primer sequences.

At the time of sacrifice, part of each fresh liver sample was frozen immediately in liquid nitrogen to preserve good quality RNA for gene expression studies. Then the samples were transferred by nitrogen tank from the surgery room and kept at −80°C until use.
Total RNA was extracted from the liver sections using AccuZol reagent according to manufacturer’s instruction. First-strand cDNA was synthesized by using 1 μg DNAse-treated RNA, 1 μL Super ScriptTM II Reverse Transcriptase (200 U), 20 pM N6 Random-Hexamer, 20 pM dNTP Mix, 4 μL 5X First-Strand buffer, 2 μL Dithiothreitol (0.1 M), and 1 μL RiboLock TMRNase inhibitor in a thermocycler (Eppendorf, Germany) at 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. Next, qRT-PCR was performed using ABI 7500 real-time PCR as follows: initial denaturation at 95°C for 10 s, 40 cycles of a two-step PCR (95°C for 5 s, 60°C for 30 s), dissociation stage at 95 °C for 15 s, 60°C for 1 min and 95°C for 15 s. The primers used are listed in table 1.

Total RNA was extracted according to the protocol of Oñate-Sánchez and Vicente-Carbajosa (2008) (link). One microgram of RNA was used as the template to produce cDNA with the TaKaRa oligo(dT) primer and M-MLV reverse transcriptase. We used the SYBR Premix Ex Taq (TaKaRa, Japan) and the ABI 7500 Real Time PCR to perform quantitative real-time PCR (qRT-PCR). AT4G34270 was used as an internal control. The qRT-PCR analysis used two technical replicates and three biological replicates. The primers used in qRT-PCR are shown in Supplementary Table S2.