Abi steponeplus pcr system

Total RNA of LV tissue was extracted with TRIzol Reagent (ThermoFisher Scientific) and was reverse-transcribed using Prime Script™ RT Reagent Kit (ThermoFisher Scientific). The PowerUP™ SYBR™ Green Master Mix kit (ThermoFisher Scientific) was used to amplify the cDNA samples with gene-specific primers [23 (link), 24 (link)] (Table 2) and collected by ABI StepOnePlus PCR system (ThermoFisher Scientific). The relative mRNA expressions of genes were calculated by the 2^-△△CT method as described previously [25 (link)].

Total RNA was isolated from cells, along with various adult tissues using the RNAiso reagent (Takara, #9109, Dlian, China) according to the manufacturer’s instruction. Purified RNA was reverse-transcribed using a HiScriptⅡ1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech, Nanjing, China). A quantitative real-time polymerase chain reaction (qPCR) was performed in a technical triplicate using SYBR Premix ExTaq II (Takara, #RR820A) with an ABI StepOnePlus PCR system (Applied Biosystem, CA, USA). All data were generated using cDNA from triplicate wells for each condition. The comparative Ct method was used to calculate the relative quantity of the target gene mRNA, normalized to bovine β-actin, and was expressed as the fold change =2^−∆∆Ct. The following procedures were used for qPCR experiments: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C, and 30 s at 60 °C. The primer sequences used for qPCR are available in Table S1.

Telomere qPCR was performed as described in previous studies [48 (link),49 (link)] with the following modifications. Genomic DNA was extracted from cells according to the manufacturer’s protocol using the TIANamp Genomic DNA Kit (TIANGEN BIOTECH, #DP304-03, Beijing, China). Primers used to amplify the telomere and the single copy reference gene β-globin for each sample are listed in Table S1. According to our results, 20 μL of the final volume per reaction contained 10 μL of 2× SYBR Premix ExTaq (Takara, #RR820A), 0.8 μL of each telomere primer (Tel1, 90 nM, Tel2, 300 nM) or 0.8 μL of each β-globin primer (150 nM), 5 μL of template DNA (7 ng/μL), and 4.4 μL double-distilled water. The Telomere and β-globin real-time amplifications were performed in the ABI StepOnePlus PCR system (Applied Biosystem, CA, USA). The Telomere qPCR conditions were 10 s at 95 °C, followed by 30 cycles of 5 s at 95 °C, 30 s at 54 °C, and 31 s at 72 °C. The relative telomere length was determined by the T/S ratio of the telomere product amplification (T), to the internal single copy reference gene β-globin (S).

Total RNA was isolated from WT and Per-KD BmN cells for qRT-PCR analysis using RNAiso™ Plus (TaKaRa, Dalian, China). The concentrations of RNA were determined and the qualities were controlled spectrophotometrically by the A260/A280 ratio (Beckman, Brea, CA, USA). Any DNA in the total RNA samples was digested with RNase-free DNase I (TaKaRa), and 1 μg of the total RNA was used to synthesize first-strand cDNA. qRT-PCR was carried out in a total reaction volume of 20 μL using an ABI StepOnePlus™ PCR system (Ambion, Foster City, CA, USA) and the fluorescent dye SYBR Premix Ex Taq (TaKaRa), according to the manufacturers’ instructions. The reaction conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s then 60 °C for 30 s. After PCR, melting curve analysis was used to confirm the amplification of specific products and the data were normalized against endogenous BmRp4962 (link)79 (link). The standard curve method was used to determine the expression levels of the samples. All the experiments were performed in triplicate. The gene-specific primers used in this study are given in Table S1.

WT and Cry1-KD cells were sampled every 4 h for 24 h for qRT-PCR. Total RNA was isolated with RNAiso™ Plus (TaKaRa, Dalian, China) from WT and Cry1-KD BmN cells, and cDNA was synthesized with the PrimeScript RT (Perfect Real Time) Reagent Kit with gDNA Eraser (TaKaRa), according to the manufacturer’s instructions. All reactions were carried out in a total reaction volume of 20 μL using an ABI StepOnePlus™ PCR system (Ambion, Foster City, CA, USA) and the fluorescent dye SYBR Premix Ex Taq (TaKaRa). Transcript levels of BmCry2, BmPer, BmTim, BmClk, and BmCyc were obtained under the following reaction conditions: 95 °C for 30 s, then 40 cycles at 95 °C for 5 s and 60 °C for 30 s. After PCR, we used a melting curve analysis to confirm the amplification of the specific products. The data were normalized with endogenous BmRp49. All the experiments were performed in triplicate. The gene-specific primers used in this study are shown in Table S1.

Quantitative reverse transcription PCR (qRT-PCR) was performed to determine the expression level of gene associated with carbohydrate metabolism. Total RNA was isolated via the hot phenol method using the TRK-1002 Kit (LC Bio, China). The resulting cDNA was stored at −20°C until qRT-PCR was performed. qRT-PCR was performed with RNA isolated from S. thermophilus MN-ZLW-002 at four different growth phases. Primers used for qRT-PCR were listed in Supplementary Table S1. qRT-PCR was carried out in 96-well plates using the ABI StepOnePlus PCR system (American). The 16S rRNA gene was used as an internal control to normalize cycle threshold (CT) values. The 2^–(ΔΔCT) method was used to assess the differences in the expression levels of sRNA genes. Three technical replicates were performed for each sample.