Moloney murine leukemia virus reverse transcriptase

Total RNA was extracted from tissue samples of wild-type and Echs1^+/- mice preserved in RNA and then converted to cDNA using random hexamers, oligo (dT) primers, and Moloney murine leukemia virus reverse transcriptase (TaKaRa). The ECHS1, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-catenin, connexin43, and myosin heavy chain β mRNA levels were measured by quantitative real-time PCR (qRT-PCR) using the ABI Prism 7900 sequence detection system (Applied Biosystems), with actin as an internal reference gene. Each reaction was performed in triplicate. The primers used are listed in Supplemental Table 2.

Total RNA was extracted using TRIzol (Invitrogen) and miRNeasy mini kit (Qiagen, Dusseldorf, Germany) according to the manufacturers’ protocols. RNA was converted to cDNA using Moloney murine leukemia virus reverse transcriptase (Takara, Dalian, P.R. China) and TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). PCR was performed using a SYBR Green PCR kit (Applied Biosystems) on a 7900HT system (Applied Biosystems). The PCR conditions were as follows: 95°C for 5 min; 35 cycles at 95°C for 5 s; 55°C for 30 s; 72°C for 30 s; and 72°C for 5 min. GAPDH and U6 were used as internal controls. Relative gene expression was calculated by the 2^−ΔΔCt method. Fold changes of gene expression were obtained by normalization with the control group. The primers used were as follows: miR-342-3p, 5′-TGCGGTCTCACACAGAAATCGCAC-3′ (forward) and 5′-CCAGTGCAGGGTCCGAGGT-3′ (reverse); U6, 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse); AEG-1, 5′-CGAGAAGCCCAAACCAAATG-3′ (forward) and 5′-TGGTGGCTGCTTTGCTGTT-3′ (reverse); cyclin D1, 5′-CCGTCCATGCGGAAGATC-3′ (forward) and 5′-GAAGACCTCCTCCTCGCACT-3′ (reverse); matrix metalloproteinase-2 (MMP-2), 5′-AGGCCAAGTGGTCCGTGTGA-3′ (forward) and 5′-TAGGTGGTGGAGCACCAGAG-3′ (reverse); GAPDH, 5′-GACTCATGACCACAGTCCATGC-3′ (forward) and 5′-AGAGGCAGGGATGATGTTCTG-3′ (reverse).

Total RNA was isolated from colon tissues using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). First-strand complementary DNA (cDNA) was synthesized from approximately 2 µg of total RNA with Moloney murine leukemia virus reverse transcriptase (Takara, Kyoto, Japan) and Oligo(dT)18 primers (Takara). qRT-PCR was performed using SuperScript™ III Platinum™ SYBR™ Green (Thermo Fisher Scientific) on a QuantStudio™ 5 Dx Real-Time PCR System. The temperature protocol was: 95 °C for 5 minutes, followed by 36 cycles of 95 °C for 15 seconds and 58 °C for 15 seconds, with a final 5-minute extension at 72 °C. The primer sequences used in the study are shown in Table 1. qRT-PCR analysis was carried out at least 3 times. Relative RNA level was calculated using the 2^-ΔΔCT method.

Two micrograms of total RNA and 0.5 pg of oligodeoxythymide were mixed in a total volume of 5 μL, incubated at 70°C for 10 min and cooled at 4°C for 2 min. The first strand buffer, 0.5 mmol each deoxynucleotide triphosphate, and 100 U Moloney murine leukemia virus reverse transcriptase (Takara) were then added into the reaction mix in a total volume of 10 μL. Reverse transcription (RT) was performed at 42°C for 90 min. In accordance with our previously established method (Cai et al., 2015 (link)), quantitative real-time RT-PCR was performed on the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA) to examine the mRNA levels of target genes in chicken tissues.