Thermoscript rt pcr kit

RNA was isolated from embryos (at least 20 embryos/sample) using the Qiagen RNeasy kit (74104). One microgram of purified RNA was used to generate cDNA using the Invitrogen Thermoscript RT-PCR kit (11146-024) and oligo-dT primers. cDNA was diluted 1:20 in nuclease-free water (Ambion). For analysis of the bad e2i2 morpholino, RT-PCR was performed and analyzed by standard agarose gel electrophoresis. For quantitative real-time PCR, three technical replicates were analyzed using an Eppendorf Realplex system. Primers were designed by Roche to be used with the Universal Probe Library. All primers used for RT-PCR and qPCR are listed in Table S1. GraphPad Prism software was used to plot the data, and error bars represent the standard error of averaged data. Statistical analyses were performed in GraphPad Prism using an unpaired student’s T test.

Total RNA was isolated using TRIZOL reagent (Invitrogen) in accordance with the manufacturer’s instructions, and the first-strand cDNA was synthesised with the Thermo Script RT–PCR kit (Invitrogen) in accordance with the manufacturer’s instructions. Applied Biosystems 7500 Real-Time PCR system software was used for real-time PCR analysis. The analysis was performed with SYBR-green I fluorescence (Applied Biosystems). Quantification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (as an internal control for gene expression in the cells) was performed with TaqMan Human GAPDH Control Reagents (with VIC Probe, Applied Biosystems). For semi-qRT–PCR analysis, RT–PCR was performed with a Platinum qRT–PCR kit (Invitrogen) in accordance with the manufacturer’s instructions. PCR products were separated on 1.5% agarose gel. The primers used in this study were as follows: ABRO1-forward-primer: 5′-TCCATAACCATCAGCCTTGTTC-3′; ABRO1-reverse-primer: 5′-CCCAATGACTTTCTTTCTCCGA-3′; GAPDH-forward-primer: 5′-GGAGCGAGATCCCTCCAAAAT-3′; GAPDH-reverse-primer: 5′-GGCTGTTGTCATACTTCTCATGG-3′; p53-forward -primer: 5′-CAGCACATGACGG AGGTTGT-3′; p53-reverse-primer: 5′-TCATCCAAATA CTCCACACGC-3′; p21-forward-primer: 5′-TGTCCGTCAGAACCCATGC-3′; p21-reverse-primer: 5′-AAAGTCGAAGTTCCATCGCTC-3′; Mdm2-forward-primer: 5′-GAATCATCGGACTC AGGTACATC-3′; and Mdm2-reverse-primer: 5′-TCTGTCTCACTAATTGCTCTCCT-3′.

Total RNA was extracted using Tri-Reagent (Molecular Research Inc., Cincinnati, OH, United States). For miRNA, the miRCURY LNA^TM Universal cDNA Synthesis kit (Exiqon Inc., Woburn, MA, United States) was used for reverse transcription of miRNA. The primers were purchased from Exiqon. The miRCURY LNA microRNA PCR SYBR Green master mix (Exiqon Inc.) was used for qPCR with the following cycle parameters: 95°C for 10 min and 40 cycles at 95°C for 10 s and 60°C for 60 s. Expression of individual microRNA was standardized to the mouse U6 gene (tissue) or miR103 (serum) (Hu et al., 2015 (link); Su et al., 2017 (link)). For mRNA we used a Thermoscript RT-PCR kit (Invitrogen, Carlsbad, CA, United States). Real-time qPCR was performed with SYBR Green PCR Reagents (Bio-Rad, Hercules, CA, United States) using the following cycle parameters: 94°C for 2 min and 40 cycles at 94°C for 15 s, 55°C for 30 s, 72°C for 30 s with final extension at 72°C for 10 min. The quantification cycle (Cq) values were defined as the number of cycles required for the fluorescence signal to exceed the detection threshold. Individual miRNA or mRNA expression was calculated as the difference between the threshold values of the two genes (2-Δcq). Melting curve analysis was routinely performed to verify the specificity of the reaction. Let-7-5p (YP00204767) was ordered from Qiagen (Germantown, MD, United States).

To examine tissue gene expression, 20–40 mg of heart tissue was homogenized using a Biospec mini bead beater. Total RNA was isolated using a Pure Link micro-to-Midi total purification kit (Invitrogen) and reverse transcribed using ThermoScript RT-PCR Kit (Invitrogen). Quantitative real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems) was used to examine gene expression.