Rnaiso plus solution

As previously described [46 (link)], total RNA was extracted with RNAiso Plus solution (TAKARA, Dalian, China, 9109), and cDNAs were synthesized using a PrimeScrip RT reagent Kit (TAKARA, RR037A). Quantitative Real-time PCR was performed on Thermo Scientific QuantStudio 3 Real-Time PCR System using FastStart Universal SYBR Green Master (Roche Life Science, Mannheim, Germany, 04913914001). The data were analyzed using the comparative threshold cycle (ΔΔCt) method and normalized to Gapdh. All PCR primers used are listed in Supplementary Table S2.

Total RNA from INS-1 cells was extracted with RNAiso Plus solution (Takara, Japan). Then, 1 ug RNA was reverse-transcribed into cDNA using PrimeScriptTM Reverse Transcriptase (Takara, Japan). Real-time PCR was conducted with the SYBR Green PCR kit (Takara, Japan). Relative expression levels of target mRNAs were normalized to β-actin and were calculated based on the 2-ΔΔCt comparative method. Primer sequences are listed in Table 2.

Expressions of IP, TP receptors, and β-actin (internal controls) were detected by real-time PCR. Vessel specimens (pooled from 2 mice for each single set of experiments) were cut open and rinsed of blood components, followed by mincing and homogenizing in an ice-cold RNAiso Plus solution (TaKaRa, Dalian, China), using a glass homogenizer. In some experiments, the opened vessel strips were further denuded of endothelium by rubbing with a moistened cotton swab, which was made around one-tip of a micro-dissecting forceps, under a binocular microscope. Total RNA was prepared according to the manufacturer’s instructions. First-strand cDNA was synthesized using total RNA (250 ng) and oligo(dT)15 primers (TaKaRa). The PCR primers for IP, TP receptors, and β-actin were described previously (19). Real-time PCR was performed using a SYBR PrimScript RT-PCR kit (TaKaRa).

Total RNA was extracted from uterine tissues with RNAiso Plus solution (TaKaRa, China). Then total RNA samples were reverse-transcribed into single-stranded cDNA in a 25 μL reaction mixture (TaKaRa, China). Quantitative PCR was performed using a SYBR Premix Ex TaqIIMix according to manufacturer’s protocol (Takara, China). The results were terminally analyzed through ABI Prism 7500 software (Applied Biosystems, USA) and housekeeping 18s mRNA level was regarded as an internal control. The primers were as follows: 18s, 5’-AATCAGGGTTCGATTCCGGA-3’ (sense) and 5’-CCAAGATCCAACTACGAG CT-3’ (antisense); prl8a2, 5’- TTATGGGTGCATGGATCACTC-3’ (sense) and 5’-CCCACGTAAGGTCATCATGGA-3’ (antisense). Melting curve analysis and agarose gel electrophoresis were performed after the quantitative PCR assays to supervise the purity of PCR products.

Total RNA was isolated from cells using a RNAiso Plus solution (TAKARA, Dalian, China, 9109), and reverse transcribed into cDNA using a PrimeScrip RT reagent Kit (TAKARA, RR037A). A real-time PCR was performed with FastStart Universal SYBR Green Master (Roche Life Science, Mannheim, Germany, 04913914001) and target gene specific primers, using a Thermo Scientific QuantStudio PCR machine. The data was analyzed using the comparative threshold cycle (ΔΔCt) method and normalized to Gapdh. All PCR primers used are listed in Table S2.

Total RNA was extracted from ovary tissues with RNAiso Plus solution (TaKaRa, China) according to the manufacturer’s protocol. RNA samples were reverse-transcribed into single-stranded cDNA in a 25 μl reaction mixture (TaKaRa, China). Real-time PCR was then performed in a 20 μl reaction volume containing 10 μl of 2x Brilliant SYBR Green Mix (TaKaRa, China), 2 μl of template cDNA, 0.5 μM primers, and 300 nM reference dye using the ABI thermal cycler 7500. The thermal cycling conditions were 95°C for 30 sec, followed by 40 cycles at 94°C for 5 sec and 60°C for 34 sec. Melting curve analysis and agarose gel electrophoresis were conducted following the quantitative PCR assays to monitor PCR product purity. The results were analyzed using ABI Prism 7500 software (Applied Biosystems, USA), and18S identified to be stable in the DEHP-treated experiment was used as an internal control. The following primers were used: 18S (ACCESSION:NR_003278): sense, 5′-AAT CAG GGT TCG ATT CCG GA--3′; antisense, 5′-CCA AGA TCC AAC TAC GAG CT-3′. StAR (ACCESSION:NM_011485): sense, 5′-CGC AGA GGT TCC ACC TGT GT-3′; antisense, 5′-TCC GGC ATC TCC CCA AA-3. CYP11A(ACCESSION:NM_019779): sense, 5′-CCG GAG CGG TTC CTT GT-3′; antisense, 5′-CCA ATG GGC CTC TGA TAA TAC TG-3′. 3β-HSD(ACCESSION:NM_013821): sense, 5′-GGA GGA AGC CAA GCA GAA AA-3′; antisense, 5′-CCC TGT GCT GCT CCA CTA GTG-3′.

Total RNA from the lung tissues was extracted by RNAiso plus solution (TaKaRa, Beijing, China). One μg of total RNA was used for synthesizing the cDNA by reverse transcription kit (Thermo Scientific, Rockford, IL, USA). SYBR Premix Ex Taq II (Takara, Beijing, China) was used for real-time PCR. Primers of α-ENaC were as follows (forward)5′-AGCCTAGAGAAG AGGACCCAG-3′ and (reverse) 5′-TCC​TCC​CGG​ACT​GTT​TGA​CT-3′. Primers of β-ENaC were as follows (forward)5′-AGCAGCTTCCTAAACAGCAGGT-3′ and (reverse) 5′-CTCACAGAT GATGCG TTTGGG-3′. Primers of γ-ENaC were as follows (forward)5′-CCCAGGCACCGA CCATTAAG-3′ and (reverse) 5′-CGT​GAA​CGC​AAT​CCA​CAA​CA-3′. All primers were obtained from Genewiz (Genewiz, Suzhou, China). PCR conditions were 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 20 s at 60°C. Gene expression was normalized with GAPDH as the housekeeping gene, and the relative expression of genes was calculated using the 2^−△△Ct method.