Cfx96 real time pcr system c1000 thermal cycler

Samples of B. cinerea-inoculated leaves were collected at 0 and 18 hpt for the qRT-PCR analysis. RNA extraction and qRT-PCR expression analyses were performed as described previously^{16 (link)}. The qRT-PCR amplification was performed using Bio-Rad CFX96 Real-Time PCR System C1000 Thermal Cycler (Bio-Rad, Hercules, California, USA) in triplicates. Reaction mixture (20 μL) contains 100 ng total RNA as a template, 10 μL 2X GoTaq qPCR Master Mix, 0.4 μL 50X GoScript RT Mix for 1-Step qRT-PCR (Promega, Madison, Wisconsin, USA), 0.3 μM each specific left and right primer (Table S1, Supporting Information). The reaction condition was as follows: 40 °C for 15 min for the reverse transcription (RT) followed by 95 °C for 10 min of RT inactivation, 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. AtActin2 was used as an endogenous reference for normalization. Expression levels were calculated by the comparative cycle threshold method, and normalization to the control was performed.

Mice from each group other than the ones used for histological analysis were weighed and then euthanized as mentioned above. The hearts were harvested and washed with PBS; HW was then recorded. The HW/BW ratio was then calculated. A small portion was cut off from the left ventricular wall and placed in a 1.5-ml tube containing 1 ml Trizol reagent (TIANGEN, Beijing, China). Total RNA was extracted from the left ventricular tissue specimen (and the cultured NRCMs) according to the protocol of the total RNA extraction kit (TIANGEN, Beijing, China). The RNA was then reverse-transcribed into complementary DNA. The CFX96 real-time PCR system C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) was used for real-time PCR. GAPDH served as the standard gene for the normalization of transcript levels of target genes. The primers used in this study were synthesized by GenScript Biotech Corp. (Nanjing, China) and the sequences are listed in Table S1.

Four weeks after the sham or TAC surgery, eight mice from each group were weighed and euthanized via carotid artery bleeding as described in Section 2.6. The heart tissues were harvested and flushed with PBS; heart weight was then recorded, and the heart weight/body weight (HW/BW) ratio was then calculated. A small piece of tissue was cut from the left ventricle and placed into a tube containing Trizol reagent. The total RNA was extracted from the specimen (or the cultured NRCMs after treatment) following the instructions of the RNA extraction kit. The RNA was then reverse-transcribed into complementary DNA using a SuperScript first-strand synthesis system (Invitrogen, CA, USA). Reverse transcriptase (RT-PCR) was performed with the CFX96 real-time PCR system-C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH served as the standard gene for the normalization of transcription levels of target genes. The sequences of primers used in this study are listed in supplementary materials Table S1.

HEV-3ra genomic RNA was quantified using SensiFAST probe No-ROX one-step kit (Bioline USA Inc.) with primers and probe listed in Table 3, following a protocol described previously (85 (link)), with some modifications. The RT-qPCR assays were performed in a CFX96 real-time PCR system, C1000 thermal cycler (Bio-Rad Laboratories). A one-step RT-qPCR thermal cycling protocol was used as follows: 45°C for 10 min and 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. In vitro-transcribed HEV-3ra RNAs were used to generate a standard curve in each RT-qPCR run, which covered a quantification range from 4 × 10² to 4 × 10⁷ RNA copies per reaction. The total RNA extracted from each of the tissue samples was diluted at 250 ng/μL, thus allowing the use of 1 μg total RNA (4 μL)/reaction as recommended in the kit. The PCR quantification data were then used to calculate the viral RNA loads as genome copies per gram of tissue. Fold increase in viral loads in pregnant rabbits (HEV-P) compared to nonpregnant rabbits (HEV-NP) was calculated as follows: (HEV RNA copies in HEV-P − HEV RNA copies in HEV-NP)/HEV RNA copies in HEV-NP.