qRT-PCR experiments were performed in vitro. SHEDs were cultured onto the developed scaffolds or directly over conventional culture plates (control group) for 0, 7, 14 or 21 days in ODM or DMEM (n = 3). The cells were then lysed with TRIzol Reagent (Invitrogen). Total RNA was extracted according to the manufacturer’s instructions, and the quantity of RNA was assessed with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). After treatment with RNase-free DNase (Invitrogen), the DNA-free RNA was used for synthesis of first-strand cDNA at 42°C for 50 min using Moloney murine leukemia virus reverse transcriptase (Invitrogen). qRT-PCR using Power SYBR Green PCR Master Mix was conducted for 45 cycles at 95°C for 15 s and at 55°C for 1 min in a 96-well format on an ABI Prism 7700 real-time PCR system (Applied Biosystems). The average Ct (threshold cycle) value for duplicate samples was used for data analysis. HMBS (hydroxymethylbilane synthase) was used to normalize the expression level of genes of interest. The primers used are shown in Table 2 .
Abi prism 7700 real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in Japan, United States
The ABI Prism 7700 Real-Time PCR system is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It enables the detection and quantification of specific nucleic acid sequences in real-time during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Sybr green reagent, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Takara Bio (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq perfect, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Fastquant rt super mix, by Tiangen Biotech (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
Superreal premix plus, by Tiangen Biotech (1 mentions)
Gotaq qpcr master mix reagent, by Promega (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
U 2900 spectrophotometer, by Hitachi (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
9 protocols using abi prism 7700 real time pcr system
1
Quantitative Real-Time PCR Analysis of SHED Cultured on Scaffolds
2
Quantifying mRNA Expression in Murine Atria
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Total RNA was extracted from mice atrial tissues using TRizol (Invitrogen, USA) and was reversely transcribed into complementary DNA (cDNA). The mRNA was amplified by RT-qPCR using SYBR Green reagent (TaKaRa, Japan) in an ABI Prism 7700 Real-Time PCR system (Applied Biosystems, USA). Primer sequences were as follows: ANGPTL4 (forward: 5′-GAG GTC CTT CAC AGC CTG CA-3′; reverse: 5′- TGG GCC ACC TTG TGG AAG AG-3′); IL-1β (forward: 5′-GCA ACT GTT CCT GAA CTC AAC T-3′; reverse: 5′-ATC TTT TGG GGT CCG TCA ACT-3′); IL-6 (forward: 5′-GTT TTC TGC AAG TGC ATC ATC G-3′; IL-6 reverse: 5′-GGT TTC TGC AAG TGC ATC ATC G-3′); collagen I (forward: 5′-GGA CAC TAC TGG ATC GAC CTA AC-3′; reverse: 5′-CTC ACC TGT CTC CAT GTT GCA-3′); collagen III (forward: 5′-CTA CCT TGC TCA GTC CTA TGA GTC TAG A-3′; reverse: 5′-TCC CGA GTC GCA GAC ACA TAT-3′); and GAPDH (forward: 5′-ACT CCA CTC ACG GCA AAT TC-3′; reverse: 5′- TCT CCA TGG TGG TGA AGA CA-3′). Experiments were performed in triplicate. The 2−ΔΔCt method was used to calculate mRNA expression. GAPDH mRNA was used as an internal control.
3
RNA Extraction and Real-Time PCR Quantification
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Total RNA from the HUEVCs was extracted using TRIzol reagent (Invitrogen, United States) [15 (link)]. Then, the purity and concentration of the RNA were detected. After that, total RNA was reverse-transcribed to cDNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser. The quantification of real-time PCR was performed using an ABI Prism 7700 Real-Time PCR System (Applied Biosystems, Foster City, CA) with the SYBR Green PCR Kit (Takara). GAPDH was used as the reference control.
4
Gene Expression Analysis of T-cell Lineage
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RNA was purified from thymus or from sorted DP, CD4+GFP+ cells, or CD4+GFP− cells. cDNA was obtain by using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Real-time PCR was performed using the ABI PRISM 7700 Real-time PCR system by using gene-specific primers and probes (Applied Biosystems). An internal control (Hprt) was used to normalize the expression value of the target gene. The primers and probes are as follows: Hprt, Mm00446968_m1; E2A, Mm01175588_m1; HEB, Mm00441699_m1; Id3, Mm00492575_m1; Foxp3, Mm00475162_m1; c-Rel, Mm01239661_m1. For human probe: ID3, Hs00954037_g1; E2A, Hs00413032_m1; HEB, Hs00918966_m1; Foxp3, Hs01085834_m1; Hprt, Hs02800695_m1. CD25 primer and probe were purchased from Roche. CD25 Probe1# from Universal Probelibrary, primer sequence: forward 5′-TGGTCTATATGCGTTGCTTAGG-3′, reverse 5′-AACTTGCTTTCTCGATTTGTCA-3′.
5
Cardiac Gene Expression Quantification
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TRizol (Invitrogen, USA) used to extract total RNA from mice atrial tissues. Reverse transcription was carried out to convert total RNA into complementary DNA (cDNA). The mRNA was amplified by Real-Time Quantitative PCR (RT-qPCR) using SYBR Green reagent (TaKaRa, Japan) in an ABI Prism 7700 Real-Time PCR system (Applied Biosystems, USA). The list of primer sequences was used as follows: BAX (forward: 5-GCC TCC TCT CCT ACT TCG G-3′; reverse: 5′-AAA AAT GCC TTT CCC CTT C-3′); BCL-2 (forward: 5′-CTC GTC GCT ACC GTC GTG ACT TCG-3′; reverse: 5′-CAG ATG CCG GTT CAG GTA CTC AGT C-3′); IL-1β (forward: 5′-CTC AAC TGT GAA ATG CCA CC-3′; reverse: 5′-GAG TGA TAC TGC CTG CCT GA-3′); TNF-α (forward: 5′-CGT CGT AGC AAA CCA CCA A-3′; reverse: 5′-GGG CAG CCT TGT CCC TTG A-3′); IL-6 (forward: 5′-TGT ATG AAC AAC GAT GAT GCA C-3′; reverse: 5′-CTG GCT TTG TCT TTC TTG TT-3′); Nrf2 (forward: 5′-AGC CCC ATT CAC AAA AGA CA-3′; reverse: 5′-GAA GTC ATC AAC AGG GAG GTT A-3′); HO-1 (forward: 5′-AAG CCG AGA ATG CTG AGT TCA-3′; reverse: 5′-GCC GTG TAG ATA TGG TAC AAG GA-3′); GAPDH (forward: 5′-TCA ACA GCA ACT CCC ACT CTT CCA-3′; reverse: 5′-ACC CTG TTG CTG TAG CCG TAT TCA-3′). Each Ct value was normalized with GAPDH to determine relative expression, and experiments were carried out in triplicate. The data were analyzed with the 2-ΔΔCT method.
6
Quantification of IL-1β Expression in NSCLC
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Total RNA was extracted from paired NSCLC and para-carcinoma tissues and NSCLC cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and 1 µg of total RNA was reverse-transcribed to cDNA using PrimeScript RT reagent kit at 37˚C for 15 min (Takara Bio, Inc.). Subsequently, qPCR was performed using SYBR Premix Ex Taq (Perfect Real Time; Takara Bio, Inc.) and an ABI Prism 7700 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used: Initial denaturation for 30 sec at 95˚C; 40 cycles of 5 sec at 95˚C and 30 sec at 60˚C; and dissociation for 15 sec at 95˚C, 30 sec at 60˚C and 15 sec at 95˚C. The specific primers for IL-1β and GAPDH were synthesized by GenScript and the following sequences were used: IL-1β forward, 5'-ATGGCAGAAGTACCTAAGCTC-3'; and reverse, 5'-TTAGGAAGACACAAATTGCATGGTGAACTCAGT-3'; GAPDH forward, 5'-TGACTTCAACAGCGACACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'. The relative expression was calculated using the 2-ΔΔCq method and presented as fold change (18 (link)). GAPDH was used for normalization.
7
Quantifying RNA Expression in Cancer
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Total RNA was isolated from tissues and T24 cells using RNAsimple Total RNA Kit (Tiangen, Beijing, China). The purity of RNA was detected at OD260/OD280, and the integrality was visualized by agarose gel electrophoresis. The reverse transcription was determined by FastQuant RT Super Mix (Tiangen). Then SuperReal PreMix Plus (SYBR Green) (Tiangen) was used to examine the miR-340, Glut-1, PCNA and Bax expression through ABI PRISM 7700 Real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the reaction condition as follows: 95˚C for 15 min, 40 cycles of 95˚C for 10 s and 60˚C for 32 s. The expression of U6 and GAPDH was detected as the normalization for miR-340 and other mRNAs, respectively. The fold change of relative expression was calculated by using 2−△△Ct method.
8
Quantitative RT-PCR Analysis of Cathepsin Genes
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The qRT-PCR was performed per a previously described method [46 (link)]. In brief, total RNA was isolated from cells using Trizol Reagent (Invitrogen, USA) and quantified. Total RNA quantity was estimated using a U-2900 Spectrophotometer (HITACHI, Fukuoka, Japan). cDNA was synthesized from 1 μg of RNA using a GoScript™ Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Real-time PCR was performed using GoTaq qPCR Master Mix reagents (Promega, Madison, WI, USA) in an ABI PRISM 7700 real-time PCR system (Applied Biosystems). RT-qPCR primer was shown as CTSA, forward: 5′-GTCGCCCAGAGCAATTTTGAG-3′; reverse: 5′-TCTCCCCGGTCAGGAAAAGTT-3′. CTSB, forward: 5′-GAGCTGGTCAACTATGTCAACA-3′; reverse: 5′-GCTCATGTCCACGTTGTAGAAGT-3′. CTSC, forward: 5′-CCAACTGCACCTATCTTGACC-3′; reverse: 5′-AAGGCAAACCACTTGTAGTCATT-3′. CTSS, forward: 5′-GCCTGATTCTGTGGACTGG-3′; reverse: 5′-GATGTACTGGAAAGCCGTTGT-3′. CTSV, forward: 5′-CGTGACGCCAGTGAAGAATCA-3′; reverse: 5′-CGCTCAGTGAGACAAGTTTCC-3′. CTSX, forward: 5′-CAGCGGATCTGCCCAAGAG-3′; reverse: 5′-CGATGACGTTCTGCACGGA-3′. GAPDH forward: 5′-CATCATCCCTGCCTCTACTG-3′; reverse: 5′-GCCTGCTTCACCACCTTC-3′. GAPDH was used as an internal control, and the difference in threshold cycle (Ct) between treated and untreated cells was determined using the 2−ΔΔCt method.
9
Quantitative RT-PCR Analysis of Inflammatory Markers
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Total cellular RNA was extracted by the TRIzol reagents. Reverse transcription was performed using the ReverTra Ace qPCR RT kit (Toyobo, Tokyo, Japan). The ABI Prism 7700 Real-Time PCR system (Applied Biosystems, Foster City, CA) was applied for the quantitative real-time reverse transcription PCR (qPCR) assay to analyze 500 ng cDNA from each sample. The mRNA primers for IL-1β, IL-6, TNF-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were based on a previous study [21] . mRNA primers for HO1, NQO1, and GCLM were provided by Dr. Huang [22] . Melting curve analysis was applied to calculate the product melting temperature [23] . We utilized the 2 -∆∆Ct method for mRNA quantification, using GAPDH as the reference gene.
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