Total RNA from aortic tissues and VSMCs was extracted using TRIzol reagent (Thermo Fisher Scientific). The isolated RNA was used to produce cDNA using a PrimeScript RT with gDNA Eraser kit (Takara). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate using SYBR1 Premix Ex Taq II (Takara). The primer sequences were as follows: TCF7L1, 5′-ACGAGCTGATCCCCTTCCA-3′ (forward) and 5′-CAGGGACGACTTGACCTCAT-3′ (reverse); αSMA, 5′-GTCCCAGACATC AGGGAGTAA-3′ (forward) and 5′-TCGGATACTTCAGCGTCAGGA-3′ (reverse); SM22α, 5′-CAACAAGGGTCCATCCTACGG-3′ (forward) and 5′-ATCTGGGCGGCC TACATCA-3′ (reverse); SRF, 5′-CGAGATGGAGATCGGTATGG-3′ (forward) and 5′-GGGTCTTCTTACCCGGCTTG-3′ (reverse); and GAPDH, 5′-AGGTCGGTGTG AACGGATTTG-3′ (forward) and 5′-GGGGTCGTTGATGGCAACA-3′ (reverse). The average cycle threshold was used to quantify relative gene expression. Relative gene expression levels were calculated by 2-ΔΔCt method.
Sybr1 premix ex taq 2
Manufactured by Takara Bio
Sourced in Japan, China
SYBR1 Premix Ex Taq II is a ready-to-use real-time PCR solution that contains SYBR1 Green I dye and Ex Taq Hot Start DNA polymerase. It is designed for quantitative gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Mircute mirna isolation kit, by Tiangen Biotech (2 mentions)
Primescript rt kit with gdna eraser, by Takara Bio (1 mentions)
Thermal cycler dice, by Takara Bio (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
8 protocols using sybr1 premix ex taq 2
1
Gene Expression Analysis in Vascular Tissues
2
Quantitative Gene Expression Analysis
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Palatal and calvarial tissues were dissected from 3 WT and 3 T1KO pups at P0 under stereomicroscopy as shown in Fig. 1F . Total RNA was isolated from the dissected tissues using the Trizol system (Nippon Gene Co., Ltd., Tokyo, Japan). First-strand cDNA was synthesized with the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Quantitative analysis of gene expression was performed by qRT-PCR using SYBR1 Premix Ex Taq II (Takara, Otsu, Japan; RR820A) and oligonucleotide primers specific for the target sequences (Supplementary Table S5 ) on a Thermal Cycler Dice (Takara). Amplification conditions were as follows: 30 s at 95 °C; 40 cycles of 95 °C for 5 s and 60 °C for 30 s; dissociation for 15 s at 95 °C; and 30 s at 60 °C. Gene expression levels were calculated relative to the levels of β-actin mRNA using the comparative Ct (2−ΔΔCt) method.
3
Gene Expression Quantification via qRT-PCR
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Total RNAs or miRNAs were extracted via Trizol reagent (Invitrogen) or miRcute miRNA isolation kit (Tiangen, Beijing, China). complementary DNAs (cDNAs) were then reverse transcribed, and quantitative reverse transcription PCR (qRT-PCR) was conducted via SYBR1 Premix Ex Taq™ II (Takara, Shiga, Japan) on 7500 Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA) with primers listed in Table 1 . U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal control.
4
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted using RNAiso Plus (TaKaRa Biotech Corporation, Dalian, China) kit and reverse transcription was performed using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa Biotech Corporation, Dalian, China). Primers for quantitative real-time PCR were designed using Primer v5.0, listed in Table 2 . PCR mixture consisted of 1 μL of cDNA, 0.8 μL of 10 pmol/μL each primer, 12.5 μL of SYBR1Premix Ex Taq™ II (TaKaRa Biotech Corporation, Dalian, China), and 9.9 μL of double-distilled H2O in a final reaction volume of 25 μL. Thermocycling parameters were 95°C for 2 min followed by 40 cycles of 95°C for 5 s, the annealing temperature for 30 s, and 60°C for 30 s.
5
Quantifying Ubiquitin-Proteasome Gene Expression
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A total of 6 ubiquitin–proteasome-related genes were selected from transcriptomic DEGs, and 12 leg muscle and cartilage samples from normal and VVD broilers were randomly selected for the test. Twelve gastrocnemius muscle samples were randomly selected from the normal broilers and VVD broilers for expression analysis of muscle atrophy-related genes. The reaction system volume was 10 µL containing 5 µL of SYBR1 Premix Ex TaqII (TaKaRa, Dalian, China), 3.2 µL of RNase-free water, 1 µL cDNA, and 0.4 µL of each primer. The qPCR was performed on a LightCycler 96 real-time fluorescent quantitative PCR instrument (Roche Applied Science, Indianapolis, IN), and the cycling conditions were as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 20 s; and 10 min extension at 72°C. The relative expression of the target gene was calculated by the 2−ΔΔCt method, and the chicken GAPDH gene was used as an internal reference gene. Three technical replicates were performed for each sample. The primer sequences are shown in Supplementary Table 1 .
6
qRT-PCR Analysis of NSCLC Samples
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Sample RNAs were extracted from NSCLC samples and adjacent normal tissues or different cell lines via Trizol reagent (Invitrogen), as well as miRNAs were extracted using miRcute miRNA isolation kit (Tiangen). The RNAs were then reverse transcribed into cDNAs using miScript Reverse Transcription kit (Qiagen). cDNAs were amplified using SYBR1 Premix Ex Taq™ II (Takara). U6 was used as the internal reference and GAPDH as the endogenous controls. Three technological replicates were used to ensure the reliability of the analysis. The primer sequences were showed: PART1, 5′‐AAGGCCGTGTCAGAACTCAA‐3′ (forward) and 5′‐GTTTTCCATCTCAGCCTGGA‐3′ (reverse); miR‐635, 5′‐TATAGCATATGCAGGGTG‐3′ (forward) and 5′‐CGCATTCGGAGTGCGAGTT‐3′ (reverse); JAK1, 5′‐GGCTCGTGCGTGTCCTAC‐3′ (forward) and 5′‐GGTCGTCCGCTTATCGTG‐3′ (reverse); JAK3, 5′‐CAGCCCCAACCAGATGTC‐3′ (forward) and 5′‐CCGCTTGATGCCTTTGTAG‐3′ (reverse); U6, 5′‐CTCGCTTCGGCAGCACA‐3′ (forward) and 5′‐AACGCTTCACGAATTTGCGT‐3′ (reverse); GAPDH, 5′‐TGTTCGTCATGGGTGTGAAC‐3′ (forward) and 5′‐ATGGCATGGACTGTGGTCAT‐3′ (reverse).
7
Quantifying Osteogenic Gene Expression
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To compare gene expression, qRT-PCR was used according to protocol. After 7 days of osteogenic induction, cells were washed with PBS and total RNA was extracted. Synthesis of cDNA was performed using SYBR1 Premix Ex Taq II (Perfect Real Time kit; TaKaRa, Japan). The primers are listed below:
GAPDH: Forward 5′- TCT GCA TCA TCC AGG AGC TTA TT−3′, Reverse 5′- TGA TAC AGA AGG CAG GTT CAC AA−3′; ALP: Forward 5′- AGC TTT CGA AGA ACA ACG GA−3′, Reverse 5′- TCT TGA AAT GCT TTG GGT CC-3′; RUNX2: Forward 5′- CCC GTG GCC TTC AAG GT−3′, Reverse 5′- CGT TAC CCG CCA AGA CAG TA-3′.
GAPDH: Forward 5′- TCT GCA TCA TCC AGG AGC TTA TT−3′, Reverse 5′- TGA TAC AGA AGG CAG GTT CAC AA−3′; ALP: Forward 5′- AGC TTT CGA AGA ACA ACG GA−3′, Reverse 5′- TCT TGA AAT GCT TTG GGT CC-3′; RUNX2: Forward 5′- CCC GTG GCC TTC AAG GT−3′, Reverse 5′- CGT TAC CCG CCA AGA CAG TA-3′.
8
Quantifying Gene Expression by qPCR
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RNA was extracted and equal amount of RNA was reverse‐transcribed to generate complementary DNA. Quantitative PCR was conducted to quantify the mRNA expression by using SYBR1 Premix Ex Taq™ II (Takara). Primers used in the study were as following: GAPDH: GGAGCGAGATCCCTCCAAAAT and GGCTGTTGTCATACTTCTCATGG; TRIM55: TGGTTTTGGATAGACATGGGGT and CTGGTGGACTCCTGCTTGTA; c‐Myc: TCCCTCCACTCGGAAGGAC and CTGGTGCATTTTCGGTTGTTG.
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