Primescript rt pcr kit

Total RNA was extracted using an RNA extraction kit (RN001, ESscience, China) according to the manufacturer’s instructions. Nanodrop2000 (Thermo Fisher Scientific Inc.) was used to measure the concentration and quality of the total RNA in samples. Complementary DNA was synthesized from 1 μg of total RNA by using the PrimeScript RT-PCR kit (RR047A, TaKaRa, Japan) and instructions provided in the manufacturer’s protocol. RT-qPCR was performed with a PrimeScript RT-PCR kit (RR820A, TaKaRa, Japan). The primers used in this study are listed in Table 1. The experiment was performed in triplicate.

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) followed by reverse transcription to generate cDNA (PrimeScript RT-PCR kit; Clontech, Palo Alto, CA, USA). The expression of transcripts was analyzed using polymerase chain reaction (PCR) with gene-specific primers. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control. The following sequence-specific primers were used in real-time PCR: hIL-19, F: 5′-CGC GGA TCC ACT CAG GAG ATG TCT GAT TTC-3′, R: 5′-AGC TGA GAA CAT TAC TTC ATG A-3′; hIL-20R1, F: 5′-AAC GCT TCT ATC CTT TCT TGG A-3′, R: 5′-GAT GAC TTT AGC CTT CCA TGC-3′; hIL-20R2, F: 5′-GTG CAC CTA GAA ACC ATG GA-3′, R': CCA TCT TCC AGA CGG AGA G-3′; hHPRT, F: 5′-TGA CCA GTC AAC AGG GGA CA-3′, R: 5′-TGC CTG ACC AAG GAA AGC AA-3′; rat IL13, F: 5′-CCA CAG GAC CCA GAG GAT ATT GA-3′, R: 5′-TAG CGG AAA AGT TGC TTG GAG TAA-3′; rat IL19, F: 5′-GCC ATG CAA ACT AAG GAC ACC-3′, R: 5′-TTG GTC ATG CAG CAC ACA TC-3′; rat GAPDH: F: 5′-ACA TGC CGC CTG GAG AAA CCT-3′, R: 5'-TCC ACC ACC CTG TTG CTG TAG-3′. The expression levels were amplified using a thermal cycler (LightCycler 480; Roche Diagnostics, Indianapolis, IN, USA) with SYBR Green I (Roche, Basel, Switzerland) for quantitative analysis normalized with GAPDH as an input control. Relative changes in mRNA expression were determined by calculating 2^−ΔΔCt.

The primers used in this study were as follows: HMOX1-specific sense primer, 5′-AGGTCCTGAAGAAGATTGCG-3′ (20 bp), HMOX1-specific anti-sense primer, 5′-GATGCTCGGGAAGGTGAAAA-3′ (20 bp); GAPDH-specific sense primer, 5′-GCACCGTCAAGGCTGAGAAC-3′ (20 bp), GAPDH-specific anti-sense primer, 5′-ATGGTGGTGAAGACGCCAGT-3′ (20 bp). Real-time quantitative RT-PCR was carried out using the PrimeScript RT-PCR Kit in combination with quantitative PCR reagent from Clontech (Mountain View, CA, USA) following the manufacturer's instructions. Samples without template and reverse transcriptase were included as negative controls, and Ct values greater than 35 were considered as negative signals. Fold changes in HMOX1 expression were calculated by comparative Ct analysis after normalization to GAPDH expression in the same samples.

Total RNA was extracted using Trizol reagent (Life Technologies no. 15596026), and cDNA was generated using a PrimeScript RT-PCR Kit (Clontech no. RR014B). qPCR was carried out using SYBR Premix Ex Taq II (Takara no. RR820L). All qPCR data represent three independent experiments, and ΔcT values were calculated using a GAPDH control. Error bars represent standard deviation, and statistical analysis was carried out by unpaired t test. Primer pairs are listed in Table S2.

Total mitochondrial RNA was isolated from myocardial tissues using Trizol (Invitrogen Inc., Carlsbad, CA, USA), and then reverse-transcribed using the RT-PCR ReverTra-Plus^TM kit (TOYOBO Co., Ltd, Tokyo, Japan) according to the manufacturer’s instructions. Semi-quantitative RT-PCR analysis of COX I and COX III mRNA expressions was performed using the Prime Script RT-PCR kit (Clontech Laboratories Inc., Mountain View, CA, USA) according to the manufacturer’s instructions and a PCR system (AG-22331; Eppendorf, Hamburg, Germany). Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as control. The primers were: COX I: forward: 5′-GGC TTC GGG AAC TGA CTT GT-3′, reverse: 5′-AAG GAT TGG GTC TCC ACC TC-3′ (annealing temperature: 60 °C; 30 cycles; PCR product: 462 bp); COX III: forward: 5′-GCC ACC ACA CCC CTA TTG TA-3′, reverse: 5′-TCC CGT TGC TAT GAA GAA TG-3′ (annealing temperature: 60 °C; 30 cycles; PCR product: 401 bp), and GAPDH: forward: 5′-TGT TCC TAC CCC CAA TGT GT-3′, reverse: 5′-CCC TGT TGC TGT AGC CGT AT-3′ (annealing temperature: 60 °C; 30 cycles; PCR product: 401 bp). The relative expression of mRNA of a specific gene to the internal control was calculated as the ratio of relative optical density using a gel imaging analyzer (EPS301; GE Healthcare, Waukesha, WI, USA) with gel analysis software (GE Healthcare, Waukesha, WI, USA).

Total RNAs were isolated using TRIzol (Catalog No.15596-026, Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. RNA was reversely transcribed to cDNA using PrimeScript RT-PCR kit (Catalog No. RR014, Takara Clontech, Tokyo, Japan) and then used for quantitative real-time PCR using SYBR Premix Ex TaqII (Catalog No. RR820, Takara Clontech) and 7500 Fast Real-Time PCR Systems (Applied Biosystems, Foster, CA, USA). MicroRNA levels were detected using PrimeScript miRNA RT-PCR kit (Catalog No.638315, Takara Clontech) according to manufacturer’s protocol. U6 and GAPDH were used as internal controls. Primer sequences for quantitative real-time PCR are listed in Table 2 and Table 3.

Total RNA was extracted using the TRI Reagent® Protocol (Sigma). RNA concentration was determined by spectrophotometry (BioPhotometer Plus, eppendorf). One microgram of total RNA was reverse-transcribed by Moloney murine leukemia virus (M-MuLV) reverse transcriptase (PrimeScript RT-PCR kit, Clontech) using oligo(dT) 12–18 as the reverse transcription primer. The reaction mixture was incubated for 50 min at 42 °C and terminated for 15 min at 70 °C.