Sybr green real time pcr master mix

According to the results of transcriptomic sequencing, five important differentially expressed genes (LEPR, STAT3, leptin, HSPA12A, and CAPN1) were selected. Premier 6.0 was used to design primers for the selected genes (Table 1) (Tiangen, Shanghai, China). β-Actin was used as an internal reference gene. The reaction system was 20 μL: 1 μL of cDNA, 0.4 μL (10 μmol/L) of upstream and downstream primers, 0.4 μL of Rox reference dye II (50x), 10 μL of SYBR green real-time PCR Master Mix (2x), and 7.8 μL of ddH₂O (Tiangen, Shanghai, China). Real-time PCR running conditions are as follows: predenaturation at 95°C for 15, 5 s at 95°C, and 34 s at 60°C, 40 cycles. The dissolution curve was analyzed after amplification. Each sample was measured in triplicate, and the average value was taken. Relative quantitative results were calculated by the 2^−ΔΔCT method (20 (link)).

Total RNA isolated from follicle granulosa tissue were extracted by using Invitrogen TRIzol reagent (Invitrogen, Carlsbad, CA, United States), following the manufacturer’s instructions. A total of 1 μg RNA was reverse transcribed into cDNA using the Fast Quant RT Kit (with gDNase) (Tiangen Biotech Co., Ltd., Beijing, China) according to manufacturer’s instructions. The expression levels of WNT4, LHCGR, HSD11B2, EDN3, EDNRB2, ADIPOQ, CD38, STAR, HSD3B1, CYP11A1, FSHR, and ER were quantified by qPCR using the SYBR Green Real-time PCR Master Mix (Tiangen Biotech Co., Ltd., Beijing, China). The primers for qPCR were designed using the Primer Premier 5 software (PREMIER Biosoft, Palo Alto, CA) and were subsequently synthesized (Sangon Biotech Co., Ltd., Beijing, China). The primer information is listed in Table 1. The cycling parameters used for qPCR amplification were as follows: initial heat-denaturation at 95°C for 4 min; 40 cycles of 95°C for 30 s, 55–60°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. A melting curve analysis was performed to exclude genomic DNA contamination and to confirm primer specificities. Gene expression was normalized using the 2^−△△CT method with the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal standard. Each biological duplicate was controlled in three technical replicates.

The cyanobacterial stock solution was added to fresh BG-11 medium (150 ml) according to the inoculation ratio of 1:100, and the culture was started. On the 3rd, 6th, 9th, 12th, and 15th day of culture, the algae were collected to measure the expression level of vp28-mOrange gene. Total RNA from the cells of mutant S. elongatus collected at different growth times (3, 6, 9, 12, and 15 days) were extracted and reverse-transcribed into cDNA using a FastQuant RT kit (Tiangen, Beijing, China). Primers (see Table S3) designed according to vp28-mOrange genes were used to quantify gene expression. The SecA gene was used as an internal reference gene to normalize the data. The reaction mixture was prepared using SYBR Green real-time PCR Master Mix (Tiangen). Final concentrations in a total volume of 25 μL were as follows: 12.5 μL of ×2 SuperRealPreMix Plus (including SYBR Green I), 0.75 μL of 10 mM forward and reverse primers, and 2 μL of cDNA, 0.5 μL×50 reference dye, and 8.5 μL RNase-free ddH₂O. The 2^−△△Ct method was used to assess the relative quantification of gene expression (25 (link)). Using an RT-qPCR system, the following PCR protocol was used: denaturation at 95°C for 15 min, 40 cycles at 95°C for 10 s, followed by 55°C for 20 s, and extension at 72°C for 30 s using an RT-qPCR system (Funglyn FTC-3000, Toronto, Canada).