Abi 7500 fast real time pcr detection system

Total RNA was extracted from the purified human sperm or rat testis tissues with TRIzol reagent (Life Technologies). Reverse transcription and polymerase chain reaction (PCR) was performed with oligo deoxythymidine (oligo-dT) primers and moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's protocol. PCR primer sequences are listed in table S2. Quantitative real-time PCR (RT-qPCR) assay was performed with GoTaq qPCR Master Mix (Promega) and an ABI 7500 Fast Real-Time PCR Detection System (Applied Biosystems). Briefly, a-20 μL PCR reaction that included 1 μL of complementary DNA, 10 μL of GoTaq qPCR Master Mix, and 0.2 μM of each primer was used and adjusted to the final volume with double distilled H₂O (ddH₂O). β-actin in parallel for each run was used as an internal control. The reactions were set up on the basis of the manufacturer's protocol. PCR conditions were incubation at 95 °C for 3 min followed by 40 cycles of thermal cycling (10 s at 95 °C, 20 s at 58 °C, and 10 s at 72 °C). The relative expression ratio of mRNA was quantified via the 2^(ΔΔCt) method 75 (link), 76 (link).

Passage 4 BMSCs from SLE patients were cultured in a 6-well plate (2.0 × 10⁵ viable cells). Total cellular RNA was extracted using Trizol reagent (Invitrogen, USA) and stored at −70°C. For quantitative RT-PCR, single-stranded cDNA was synthesized in a 200 μL reaction volume containing 30 μL total RNA using PrimerScript RT reagent Kit (TaKaRa, China). Quantitative RT-PCR was performed on an ABI 7500 FAST real-time PCR detection system (Applied Biosystems, USA) using SYBR Green detection mix (Takara, China). StepOne Software v2.1 was used to detect the mix, and the thermal profile for RT-PCR was 95°C for 30 seconds, followed by 40 cycles of 5 seconds at 95°C with 34 seconds at 60°C. The specific primers (TaKaRa, China) used are shown in Table 2.
Relative expression of the target genes was calculated with the 2^−ΔΔCt method. Briefly, a value for the cycle threshold (Ct) was determined for each sample and defined as the mean cycle at which the fluorescence curve reached an arbitrary threshold. The ΔCt for each sample was then calculated according to the formula Ct target gene − Ct GAPDH; ΔΔCt values were then obtained by subtracting the ΔCt of a reference sample (average ΔCt of the control group) from the ΔCt of the studied samples. Finally, the expression levels of the target genes in the studied samples as compared with the reference sample were calculated as 2^−ΔΔCt.

Total RNA was extracted using TRIzol Reagent according to the manufacturer’s protocol (Invitrogen, Beijing, China), and cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio, Otsu, Japan). mRNA expression was examined by real-time PCR using FastStart Universal SYBR Green Master Mix (Roche, Mannheim, Germany) with gene-specific primers and the ABI 7500 Fast Real-time PCR Detection System (Applied Biosystems, Foster City, CA, USA). The results were normalized to the expression of GAPDH. The primer sequences were as follows: EPB41L4A-AS2-F: 5′-CGGAGCAGGTGCAATCTGT-3′; EPB41L4A-AS2-R: 5′-CCCTCGTGTCTCCCCTAACTG-3′; RARRES1-F: 5′-AAACCCCTTGGAAATAGTCAGC-3′; RARRES1-R: 5′-GGAAAGCCAAATCCCAGATGAG-3′; ST18-F: 5′-CAAACCACCTAGAGTCCCAAAG-3′; ST18-R: 5′-ACACCTGTTCTCACAAGGGATA-3′; BMP4-F: 5′-CAAACCACCTAGAGTCCCAAAG-3′; BMP4-R: 5′-GACGGCACTCTTGCTAGGC-3′; FOXA2-F: 5′-GGAGCAGCTACTATGCAGAGC-3′; FOXA2-R: 5′-CGTGTTCATGCCGTTCATCC-3′; FOXL1-F: 5′-GCCTCGCCCATGCTGTATC-3′; FOXL1-R: 5′-CGTTGAGCGTGACCCTCTG-3′; LRIG1-F: 5′- GGACTTGCCGAACCTACAGG-3′; LRIG1-R: 5′-GCTGCGAATCTTGTTGTGCTG-3′; RASSF1-F: 5′-ATGTGCCTACCTGATACTTT-3′; RASSF1-R: 5′-ATGGTGAACCTGGAGAAC-3′; GAPDH-F: 5′-CATGTTCGTCATGGGTGTGAA-3′; and GAPDH-R: 5′-GGCATGGACTGTGGTCATGAG-3′.

Total RNA was extracted from leaves of N. benthamiana according to the method of Wu et al. (2017) (link) using a Plant RNA Kit (Omega Bio-Tek, United States). First-strand cDNA was synthesized using reverse transcriptase (TaKaRa Bio Inc., Dalian, China) and oligo(dT) primers. Reverse transcription PCR (RT-PCR) products were examined by agarose gel electrophoresis. Quantitative RT-PCR (qRT-PCR) was performed using SYBR Premix Ex Taq (TaKaRa) and an ABI 7500 Fast Real-Time PCR Detection System (Applied Biosystems, United States). PCR reactions were heated to 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 60°C for 30 s. Gene expression in each sample was normalized to expression of N. benthamiana EF-1α, and relative expression levels calculated by the 2^-ΔΔC_T method (Livak and Schmittgen, 2001 (link)). Gene-specific PCR primers shown in Table 1 are same for RT-PCR and qRT-PCR analysis.

Total RNA was isolated using Plant Spectrum Total RNA isolation kit according to manual instructions (Sigma –Aldrich, http://www.sigmaaldrich.com). After DNaseI treatment (Ambion Inc, Austin TX USA), RNA integrity was checked by electrophoresis and quantified by using using a NanoDrop^® ND-1000 UV-Vis spectrophotometer. 2 μg RNA was reverse transcribed using oligo(dT) primers and Superscript II RT (Invitrogen, Rockville, MD, USA) into first-strand c-DNA in a 20-μL reaction as per manual instructions. Quantitative real-time PCR was performed by Express SYBR^®Green ER™ qPCR SuperMix Universal (Invitrogen) using the ABI 7500 Fast Real-Time PCR Detection System (Applied Biosystems). The gene-specific primers listed in Table S1 were utilized. UBQ10 was taken as an internal control. Relative expression was calculated from threshold cycle values52 (link)). Three independent qRT-PCR reactions were performed on different cDNA samples.