Quantitative RT-PCR (qRT-PCR) was performed on isolated mRNA using TaqMan probe based chemistry and an ABI Prism 7900HT fast Sequence Detection System (Life Technologies), as previously described [7 (link)]. All primer/probe sets were from Applied Biosystems, Life Technologies.
Abi prism 7900ht fast
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7900HT/FAST is a real-time PCR system designed for high-throughput gene expression and quantification analysis. It features a 384-well block format and can perform fast cycling for rapid results. The system is equipped with a sensitive optical detection system and compatible with a wide range of fluorescent dyes and probes.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Vazyme (2 mentions)
Taqman master mix and primers, by Thermo Fisher Scientific (1 mentions)
Qscript cdna supermix, by Quanta Biosciences (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Transscript all in one first strand cdna synthesis kit, by Transgene (1 mentions)
Trizol reagent, by Transgene (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
11 protocols using abi prism 7900ht fast
1
Quantitative Real-Time PCR Analysis
2
Quantitative Real-Time PCR Analysis
3
Quantification of Rat P4/P5-ATPase mRNA
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Total mRNAs were isolated from rat livers using a protocol previously described by Eloranta et al. [17 (link)]. Double stranded complementary DNAs (cDNAs) of rat P4-ATPases (Atp8a1, Atp8b1, Atp9a, Atp11a1, Atp11c), and a P5-ATPase (Atp13a1) were amplified from total mRNA using TaqMan master mix and primers (Life Technologies, Portland, CA, USA) (S1 Table ) by reverse transcription—polymerase chain reaction (qRT-PCR). Transcript levels were measured using an ABI Prism 7900HT fast qRT-PCR system (Applied Biosystems, Carlsbad, CA, USA). Measured transcript values were normalized against the rat β-actin and GAPDH cDNA levels using the comparative threshold cycle method.
4
Quantitative Real-Time PCR of Liver Transcripts
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RNA was extracted from frozen liver tissues (~50 mg) using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 µg of total RNA using qScript cDNA SuperMix (Quanta Biosciences), and the products were diluted to 1:10 before use in subsequent reactions. Gene-specific primers were used in each reaction mixture, and all results were normalized to the ribosomal protein β-actin mRNA (primer sequences can be found in Table S1 in the supplemental material). Quantitative PCR (QPCR) assays were carried out using SYBR green QPCR master mix with an ABI Prism 7900HT Fast real-time PCR sequence detection system (Applied Biosystems). The reactions products were analyzed with the ΔΔCT method.
5
Quantifying m6A-Modified YAP Transcripts
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Reverse transcription was performed on 10 μl m6A PolyA+ RNA from the MeRIP with the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). After diluting cDNA two-fold, quantitative real-time PCR was performed using the ABI Prism 7900HT/FAST (Applied Biosystems, USA) and primers from Integrated DNA Technologies, Inc. (Coralville, Iowa). Primers used are listed in the follows. Primer efficiency was verified to be over 95% for all primer sets used. Quantification of mRNA from the MeRIP was carried out via 2-ΔΔct. analysis against non-immunoprecipitated input RNA. All real-time PCR primer sets were designed so the products would span at least one intron (> 1 kb when possible), and amplification of a single product was confirmed by agarose gel visualization and/or melting curve analysis. Primers for MeRIP:
YAP m6A peak1 Forward primer: 5′-TGCGCGTCGGGGGAGGCAGAAG-3′.
YAP m6A peak1 Reverse primer: 5′-GGAATGAGCTCGAACATGCTG-3′.
YAP m6A peak2 Forward primer: 5′-TGAACCAGAGAATCAGTCAGAG-3′.
YAP m6A peak2 Reverse primer: 5′-GTACTCTCATCTCGAGAGTG-3′.
YAP m6A peak3 Forward primer: 5′-CCAGTGTCTTCTCCCGGGATG-3′.
YAP m6A peak3 Reverse primer: 5′-TATCTAGCTTGGTGGCAGCC-3′.
YAP m6A peak1 Forward primer: 5′-TGCGCGTCGGGGGAGGCAGAAG-3′.
YAP m6A peak1 Reverse primer: 5′-GGAATGAGCTCGAACATGCTG-3′.
YAP m6A peak2 Forward primer: 5′-TGAACCAGAGAATCAGTCAGAG-3′.
YAP m6A peak2 Reverse primer: 5′-GTACTCTCATCTCGAGAGTG-3′.
YAP m6A peak3 Forward primer: 5′-CCAGTGTCTTCTCCCGGGATG-3′.
YAP m6A peak3 Reverse primer: 5′-TATCTAGCTTGGTGGCAGCC-3′.
6
Colon Cancer Tissue RNA Extraction
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The tissue specimens of 16 COAD patients were collected from the Department of General Surgery, First Affiliated Hospital of Anhui Medical University. Experiments using patients’ specimens were approved by the Institutional Ethics Committee, First Affiliated Hospital of Anhui Medical University. Total RNAs were extracted from colon cancer tissues and cells using a total RNA Quick Extraction Kit (Generay Biotech, Shanghai, China) in accordance with the manual. Then, the extracted RNAs were reverse-transcribed into cDNAs using the PrimeScript™ RT Master Mix (TaKaRa, Dalian, Liaoning, China). qRT-PCR was performed with TB Green™ Premix Ex Taq™ II (Takara, Dalian, Liaoning, China) in ABI Prism 7900HT/FAST (Applied Biosystems, USA). The relative expression levels were analyzed via the 2−ΔΔCT method normalized by GAPDH expression. The Wilcoxon test was used to analyze the differences in gene expression between the cancer and normal samples. t-test was used to evaluate the differences in gene expression between cells treated without or with NEC-1 and TNFα. The primer sequences used in our study are shown in Supplementary Data Sheet S2
7
Extraction and Quantification of Total RNA
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Total RNA was extracted from villi or cells with Trizol (Invitrogen, Carlsbad, USA); nuclear and cytoplasmic RNA was prepared using a PARIS™ Kit (Invitrogen Carlsbad, USA). We used 20 URPL patients and 20 controls in the cohort for the validation. First strand cDNA was synthesized (Vazyme, Nanjing, China) and quantitative PCR was performed. All reactions were performed in triplicate with SYBR Green master mix (Vazyme, Nanjing, China) under the following conditions: 5 min at 95 °C for initial denaturation, followed by 40 cycles of segments of 95 °C for 30 s and 60 °C for 30 s in the ABI Prism 7900HT/FAST (Applied Biosystems, Foster City, USA). The expression levels of GAPDH were used to normalize the expression levels of the tested genes. All the primer sequences used were listed in Supplementary Material, Table S2.
8
Quantification of Gene Expression by RT-qPCR
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Total RNA was isolated from snap-frozen tissue samples using TRIzol reagent (Invitrogen) according to the manufacturer’s guidelines. 0.5μg of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems) following the manufacturer’s instructions. cDNA was analyzed by Real Time Quantitative PCR (ABI PRISM 7900HT FAST or Quant Studio 6 Real-Time PCR system, Applied Biosystems) using the PowerUp SYBR Green master mix (Applied Biosystems #A25742) and primers were designed according to Harvard validated PrimerBank. Relative mRNA levels were calculated using the 2-ΔΔCT method and normalized to GAPDH mRNA. For human muscle biopsies, total RNA was extracted from approximately 20 mg of rectus abdominal muscle using TRIzol (Invitrogen). 1 μg of RNA was reverse transcribed using the SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Gene expression was analyzed by qRT-PCR (Quant Studio 5 Real-Time PCR system, Applied Biosystems) using the PowerUp SYBR Green master mix (Applied Biosystems #A25742). Data were normalized to beta-actin gene expression. All RT-qPCR primers are listed in Table S1 .
9
Quantitative Real-Time PCR for Gene Expression
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Total RNA was isolated from samples using Trizol reagent (TransGen Biotech, Beijing, China). RNA was reverse transcribed to cDNA using TransScript All-in-One - First-strand cDNA Synthesis Kit (TransGen Biotech). Using human GAPDH gene as the internal control, cDNA was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using Applied Biosystems (USA). The final qPCR reaction mixture contained 10 μ L of Bestar® SYBR Green QPCR Master Mix. The amplification steps were as follows: denaturation at 94 °C for 5 min, amplification at 94 °C for 30s, amplification at 58 °C for 30s and amplification at 72 °C for 30s, 40 cycles in total. The reaction was stopped at 25 °C for 5 min. ABI Prism 7900HT/FAST (Applied Biosystems, USA) was used to detect and analyze its relative expression, calculated using the formula 2−ΔΔct The RT-PCR primers were as follows. Reaction PCR primers (Supplementary Material 1
10
Quantitative Analysis of lncRNAs
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TRIzol reagent (Invitrogen, Carlsbad, USA) was used to extract the total RNA from decidual tissues. Quantitative real-time PCR was used to examine the expression levels of lncRNAs and genes. The primer sequences are listed in Table S1 . Using SYBR green master mix (Vazyme, Nanjing, China), quantitative real-time PCR reactions were performed by the ABI Prism7900HT/FAST (Applied Biosystems, Foster City, USA). Gene expression was normalized to the internal control (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). For quantification, the relative CT method (2-ΔΔCt method) was employed.
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