Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific). After RNase-free DNAse I (Takara, Tokyo, Japan) treatment, DNA-free RNA was reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The expression level of NEAT1 was determined using the SYBR Green PCR kit (Thermo Fisher Scientific) and GAPDH served as an endogenous control. miR-34a-5p expression was examined using TaqMan miRNA assay (Thermo Fisher Scientific) and U6 snRNA was used as the internal control. All PCR steps were performed on the ABI 7300 system (Thermo Fisher Scientific) and the relative gene expression was calculated using the 2−∆∆CT method. The primers were as follows: NEAT1, 5′-CTTCCTCCCTTTA ACTTATCCATTCAC-3′ (forward) and 5′-CTCTTCCTC CACCATTACCAACAATAC-3′ (reverse); miR-34a-5p, 5′-TGGCAGTGTCTTAGCTGGTTGT-3′ (forward) and 5′-GCGAGCACAGAATTAATACGAC-3′ (reverse); GAPDH, 5′-ACATCAAGAAGGTGGTGAAGCAGG-3′ (forward) and 5′-CTCTTGCTCTCAGATCCTTGCTGG-3′ (reverse); U6, 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse).
Abi 7300 system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, China, Germany, United Kingdom
The ABI 7300 system is a real-time PCR instrument designed for gene expression analysis and quantification. It employs the Taqman® technology and can be used to detect and quantify specific DNA sequences in real-time during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (82 mentions)
Trizol, by Thermo Fisher Scientific (26 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (15 mentions)
Sybr premix ex taq, by Takara Bio (14 mentions)
Trizol reagent, by Takara Bio (13 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (11 mentions)
Sybr premix ex taq kit, by Takara Bio (9 mentions)
Sybr select master mix, by Thermo Fisher Scientific (9 mentions)
Primescript rt master mix, by Takara Bio (9 mentions)
Primescript rt reagent kit, by Takara Bio (9 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (9 mentions)
Dnase 1, by Roche (8 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (8 mentions)
Sybr green qpcr master mixes, by Thermo Fisher Scientific (8 mentions)
Trizol kit, by Thermo Fisher Scientific (7 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (6 mentions)
Qpk 201 sybr green master mix, by Toyobo (6 mentions)
Rnaiso plus, by Takara Bio (6 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
243 protocols using abi 7300 system
1
Quantification of NEAT1 and miR-34a-5p
2
Quantifying Tendon Gene Expression
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Laser capture microdissection collects very small amounts of tissue which is not adequate for mRNA expression analysis and therefore whole tendon was used for the mRNA expression analysis. RNA extraction from whole SDFT and CDET was carried out followed by reverse transcription. Quantitative real-time PCR (qRT-PCR) was performed on an ABI7300 system (Thermo Fisher Scientific Waltham, Massachusetts) using the Takyon ROX SYBR 2X MasterMix (Eurogentec, Liege, Belgium). qRT-PCR was undertaken using previously validated gene-specific primers for DCN, FMOD, BGN, COMP, COL1A1, COL1A2, COL3A1, TGFB1, and GAPDH as a reference gene (Peffers et al., 2013 (link); Taylor et al., 2009 (link); Supplementary file 2 ). Relative expression levels were normalised to GAPDH expression and calculated with the formula E−ΔCt following primer efficiency calculation.
3
Quantifying LINC00265 Expression in Tissues and Cells
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Total RNA of tissues and cells was extracted by TRIzol reagent (Invitrogen), followed by
detection of purity and reverse transcription into cDNA. Then, qRT-PCR was performed with
SYBR®Premix-Ex-Taq™ (Takara, Japan) on the ABI7300 system (Thermo-Fisher
Scientific, USA). Relative expression of LINC00265 was calculated by 2-ΔΔCtmethod using GAPDH as the internal reference. The utilized primer sequences are as
following:
LINC00265-
F: 5ˊ-GGAAGAGAGACTGACTGGGC-3ˊ
R: 5ˊ-GTTTCGCTGTCACCCCTCTG-3ˊ
GADPH-
F: 5ˊ-GTCAACGGATTTGGTCTGTATT-3ˊ
R: 5ˊ-AGTCTTCTGGGTGGCAGTGAT-3ˊ
detection of purity and reverse transcription into cDNA. Then, qRT-PCR was performed with
SYBR®Premix-Ex-Taq™ (Takara, Japan) on the ABI7300 system (Thermo-Fisher
Scientific, USA). Relative expression of LINC00265 was calculated by 2-ΔΔCtmethod using GAPDH as the internal reference. The utilized primer sequences are as
following:
LINC00265-
F: 5ˊ-GGAAGAGAGACTGACTGGGC-3ˊ
R: 5ˊ-GTTTCGCTGTCACCCCTCTG-3ˊ
GADPH-
F: 5ˊ-GTCAACGGATTTGGTCTGTATT-3ˊ
R: 5ˊ-AGTCTTCTGGGTGGCAGTGAT-3ˊ
4
Quantitative Expression Analysis of MicroRNAs and Genes
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Total RNA in tissue was extracted using Trizol kits (Invitrogen, CA, USA), and the reverse transcription was performed using high-power capacity cDNA reverse transcription kits and TaqMan MicroRNA reverse transcription kits (Thermo Fisher Scientific, MA, USA). The PCR was conducted by the ABI 7300 system (Thermo Fisher Scientific) and SYBR Premix Ex Taq (TaKaRa Biotechnology Co., Ltd., Liaoning China). The primers are shown in Table 1 and data were analyzed by 2−ΔΔCt method.
Primer sequence
| Gene | Primer sequence (5′-3′) |
|---|---|
| MiR-125b-5p | Forward: 5′-GTGCTAGCACTGGACCACCTGTTTGC-3′ |
| Reverse: 5′-GTCTGCAGAAGGGTGTATTACCATCACTTC-3′ | |
| U6 | Forward: 5′-TTAGCATGGCCCCTGC-3′ |
| Reverse: 5′-TGCGTGTCGTGGAGTC-3′ | |
| TOP2A | Forward: 5′-GCCATTGGCTGTGGTATTG-3′ |
| Reverse: 5′-GAGAAGCTTCTCGAACATTGAG-3′ | |
| VEGF | Forward: 5′-ACATTGGCTCACTTCCAGAAACAC-3′ |
| Reverse: 5′-GGTTGGAACCGGCTCATCTTTATC-3′ | |
| β-actin | Forward: 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ |
| Reverse: 5′-ATGGAGCCACCGATCCACA-3′ |
MiR-125b-5p microRNA-125b-5p, TOP2A topoisomerase II alpha, VEGF vascular endothelial growth factor
5
Quantitative Analysis of Vwf mRNA
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Total RNA was isolated from the spleen and reverse transcribed. Real‐time polymerase chain reaction (RT‐PCR) was performed using the ABI 7300 system (Thermo Fisher Scientific) with TaqMan probes for mouse Vwf messenger RNA (mRNA), and for 18S ribosomal RNA as an internal control. Each threshold cycle was obtained, and the double‐delta threshold cycle method was used to calculate the expression values.
6
RNA Isolation and qPCR Analysis of RAW 264.7 and Aorta Samples
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Total RNA from RAW 264.7 cells or aortas was isolated using an RNeasy minikit (74104; Qiagen, Venlo, the Netherlands). RNA (1 μg) was used to synthesize cDNA by reverse transcription with an RT2 first-strand kit (330401; Qiagen). Real-time PCR analysis was performed on an ABI 7300 system (Thermo Fisher Scientific) with RT2 first SYBR green/ROX PCR master mix (330530; Qiagen). Specific primers for each gene were purchased from Qiagen. Genes of interest were normalized to mouse β-actin.
7
B16-F10 Cell Total RNA Isolation and Analysis
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Total RNA from B16-F10 cells was isolated using an RNeasy minikit
(74104; Qiagen, Hilden, Germany). By reverse transcription with an
RT2 first-strand kit (330401; Qiagen, Hilden, Germany), RNA (1
μg) was used to synthesize cDNA. Real-time PCR analysis was performed on
an ABI 7300 system (Thermo Fisher Scientific, Waltham, MA) with RT2first SYBR green/ROX PCR master mix (330530; Qiagen, Hilden, Germany). Specific
primers for each gene were purchased from Qiagen. Genes of interest were
normalized to mouse β-actin.
(74104; Qiagen, Hilden, Germany). By reverse transcription with an
RT2 first-strand kit (330401; Qiagen, Hilden, Germany), RNA (1
μg) was used to synthesize cDNA. Real-time PCR analysis was performed on
an ABI 7300 system (Thermo Fisher Scientific, Waltham, MA) with RT2first SYBR green/ROX PCR master mix (330530; Qiagen, Hilden, Germany). Specific
primers for each gene were purchased from Qiagen. Genes of interest were
normalized to mouse β-actin.
8
Transcriptional Profiling of EPCP Cells
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Gene expression in sorted EPCPs (c‐kit+dim/EpCAM+ cells) were compared with isolated c‐kit−/EpCAM+ epithelial cells. RNA was isolated by using a RNeasy Mini kit (catalog no. 74104; SABiosciences, Qiagen, Valencia, CA,
http://www.qiagen.com ); cDNA was made by using an RT2 First Strand Kit (catalog no. 330401; SABiosciences, Qiagen) and applied to the Mouse Stem Cell Transcription Factors RT2 Profiler Array (Qiagen, PAMM‐501Z). Quantitative reverse‐transcriptase polymerase chain reaction was performed on the ABI 7300 system (Thermo Fisher), and data were analyzed by using online normalization and analysis tools (provided in the public domain,
http://sabiosciences.com/pcrarraydataanalysis.php ). Three independent experiments were performed.
9
Quantitative PCR Gene Expression Analysis
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Total RNA extraction, first-strand cDNA synthesis and qRT-PCR were performed following a published protocol [55 (link)]. Approximately 20 larvae (n = 4 replicates) were homogenized on ice to extract the total RNA with 1 mL of TRIzol reagent. The total RNA concentration and absorbance at 260/280 nm ratios were determined with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The reverse transcription of RNA was carried out with a PrimeScript® RT Reagent kit (Yeasen, Shanghai, China) following the manufacturer’s instructions. qRT-PCR was analyzed on an ABI 7300 system (Thermo Fisher Scientific) with the SYBR® Real-time PCR Master Mix-Plus kits (Yeasen, Shanghai, China). The PCR program was as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, 66 °C for 20 s and 72 °C for 20 s. The primer sequences of the genes in this study were designed using the online Primer 3 software (http://primer3.ut.ee/ (accessed on 6 September 2022)) and are listed in Table S1 . In our study, β-actin was used as the reference gene, as its transcription was stable upon trichlorfon exposure. The relative change in the target gene transcription level was calculated using the 2−ΔΔCt method.
10
Cardiac Gene Expression Profiling
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The expression of ANP, nuclear factor of activated T-cells (NFAT), α-actinin, β-myosin heavy chain (MHC), TRPC3 and TRPC6 at the mRNA level was determined by RT-qPCR assay. Total RNA was obtained by TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA concentration was measured by a Nanodrop 2000 (Thermo Fisher Scientific, Inc.). Then, cDNA was synthesized from the total RNA with PrimeScript RT Mix Kit (Takara Bio, Inc.) at 37°C for 15 min and then 85°C for 5 sec. The cDNA was amplified with SYBR Green PCR Mix (Thermo Fisher Scientific, Inc.) and an ABI 7300 system (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation, 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The expression levels were normalized to that of GAPDH. The primers for qPCR are as follows: TRPC3-forward (F), 5′-TGTGGTCTGAGTGCAAGGAG-3′ and TRPC3-reverse (R), 5′-ACCTCTGGTGGGAGTGTGAC-3′; TRPC6-F, 5′-TTTGCTGAAGGCAAGAGGTT-3′ and TRPC6-R, 5′-TTGTTTCTGGCTGCATTCTG-3′; ANP-F, 5′-ATACAGTGCGGTGTCCAACA-3′ and ANP-R, 5′-CGAGAGCACCTCCATCTCTC-3′; brain natriuretic peptide (BNP)-F, 5′-GGAAATGGCTCAGAGACAGC-3′ and BNP-R, 5′-CGATCCGGTCTATCTTCTGC-3′; β-MHC-F, 5′-CCTCGCAATATCAAGGGAAA-3′ and β-MHC-R, 5′-TACAGGTGCATCAGCTCCAG-3′; GAPDH-F, 5′-CTCATGACCACAGTCCATGC-3′ and GAPDH-R, 5′-TTCAGCTCTGGGATGACCTT-3′.
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