Total RNA extraction from the lumbar nucleus pulposus specimens and cells was performed using an RNA extraction kit (Total RNA Purification kit; Sigma Aldrich; Merck KGaA). Total RNA was reverse transcribed into cDNA using the TaqMan® MicroRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RT reaction conditions were as follows: 37°C for 60 min, followed by 85°C for 5 min and 4°C. The cDNA samples were subjected to real-time PCR using a SYBR Green One-Step RT-qPCR kit (Thermo Fisher Scientific, Inc.) on an ABI 7900HT instrument (ABI; Thermo Fisher Scientific, Inc.). The qRCR reaction conditions were as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 10 sec, 58°C for 30 sec and 72°C for 30 sec. The primer sequences for miR-222 were as follows: Forward primer: 5′-AGCUACAUCUGGCUACUGGGU-3′ and reverse primer: 5′-CCAGUAGCCAGAUGUAGCUUU-3′. The primer sequences for U6 were as follows: Forward primer: 5′-CTCGCTTCGGCAGCACAT-3′; reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′. All samples were tested in triplicate. The relative expression was measured using the 2−ΔΔCq method (21 (link)). Gene expression results were normalized by the internal control U6.
Abi 7900ht instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The ABI 7900HT instrument is a real-time PCR system designed for high-throughput gene expression analysis and genotyping. It features 96-well and 384-well sample block formats and utilizes Sequence Detection System software for data collection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Primescript rt reagent kit, by Takara Bio (5 mentions)
Abi sequence detector software version 2, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Sybr premix ex taq, by Takara Bio (4 mentions)
Taqman gene expression assay kit, by Thermo Fisher Scientific (3 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Miscript sybr green pcr kit, by Qiagen (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Reverse transcription assay kit, by Thermo Fisher Scientific (2 mentions)
Sds software v2, by Thermo Fisher Scientific (2 mentions)
Rq manager software, by Thermo Fisher Scientific (2 mentions)
Primerscriptrt reagent, by Takara Bio (2 mentions)
Sybr green detection system, by Takara Bio (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Total rna purification kit, by Merck Group (1 mentions)
Taqman microrna rt kit, by Thermo Fisher Scientific (1 mentions)
69 protocols using abi 7900ht instrument
1
Quantifying miR-222 Expression in Lumbar Nucleus Pulposus
2
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using a NucleoSpin miRNA kit (Macherey-Nagel). cDNA was generated using a High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific). In addition, cDNA obtained from HCC tissue explants was also pre-amplified using the TaqMan® Preamp Master Mix Kit (Thermo Fisher Scientific). cDNA was used to perform real-time PCR (qRT-PCR) with TaqMan® gene expression assays and the primers/probes reported in Supplementary Table S2. qRT-PCR assays were conducted using an ABI 7900HT instrument (Thermo Fisher Scientific). Data analysis was performed with SDS 2.2.2 software (Thermo Fisher Scientific).
3
Upland Cotton Drought Stress Response
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Upland cotton seeds (CCRI24 variety) were obtained from the Institute of Cotton Research of the Chinese Academy of Agricultural Science. Seed germination was performed on a wet filter paper at 28°C for 3 days, after which the seeds were transferred to liquid culture medium (Yang et al., 2014 (link)). At the three-leaf stage, the seedlings were treated with 20% PEG6000. The roots were sampled at 0, 1, 3, and 6 h post-treatment, immediately frozen in liquid nitrogen, and stored at −80°C. In addition, the true leaf, stem, and root under normal conditions were sampled, immediately frozen in liquid nitrogen, and stored at −80°C. Total RNA was purified from the samples using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China), following the manufacturer's recommended protocol. The first strand of cDNA was synthesized using the PrimeScript® RT Reagent Kit (Takara, Dalian, China). Real-time PCR amplifications were performed in an ABI 7900HT instrument (Thermo Fisher, USA) using SYBR Premix Ex Taq™ II (Takara, Dalina, China). Upland cotton histone 3 mRNA (GenBank accession number AF024716 ) was used as an internal control. The dissociation curves of each reaction were assessed, and the cycle threshold (CT) 2−ΔΔCT method was used to calculate the expression level of each target gene as previously reported (Gong et al., 2017a (link)).
4
RNA Extraction and qPCR Analysis
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Extraction of total RNA from peripheral blood was conducted with TRIzol (cat. no. 15596-028; Beijing Solarbio Science & Technology Co., Ltd.) and RNA was converted to cDNA using a reverse transcription kit (cat. no. R202; EnzyArtisan Biotech Co., Ltd.) according to the manufacturer's instructions. Subsequently, qPCR was conducted utilizing 2X S6 Universal SYBR qPCR Mix (cat. no. Q204; Xinbei) on an ABI 7900HT instrument (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for 3 sec, and annealing and extension at 60˚C for 10 sec. β-actin acted as an internal control. The primer sequences are listed in Table I . The mRNA fold variation was calculated with the 2-ΔΔCq method (25 (link)).
5
qRT-PCR Assay for Gene Expression
6
Quantification of Metabolic Gene Expression
7
Quantitative PCR of p38 in Liver
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Real-time quantitative PCR (qRT-PCR) was performed on p38 in the liver tissue of NL, AR, and HY. Each group had three replicates and β-actin was used as a quantitative reference gene. The primers were designed by Primer Premier 5.0 (Premier, Markham, ON, Canada; Table 1 ). Real-time PCR was performed on an ABI 7900HT instrument (Applied Biosystems, Foster City, CA, USA). The qRT-PCR reaction volume was 20 μL: 10 μL of ChamQ SYBR qRT-PCR Master Mix (Vazyme, Nanjing, China), 0.4 μL of each forward and reverse primer (10 μM), 1.0 μL of cDNA template, and 8.2 μL of RNase-free water. The reaction procedure was set as follows: initial denaturation at 95 °C for 30 s; 95 °C for 10 s; 60 °C for 30 s; 40 cycles. This was followed by a melting curve analysis (95 °C for 10 s, 60 °C for 60 s, and 95 °C for 15 s). The transcript levels of the target gene were analyzed by the 2−ΔΔCt method. All the data are expressed as mean ± SD, with p < 0.05 representing significant differences.
8
Optimizing Allele Frequency Profiling
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Custom TaqMan assays (primer and reporter sequences in Supplementary Table S2 ) were designed to determine the allele frequency of seven variants identified by WES, in a larger cohort of 193 unrelated indigenous African probands with IRDs but no known causative mutation. In order to determine the optimal template concentration, two control samples were screened for each assay (including a positive control for each), at 8, 6, 4, 3, 2, and 1.5 ng/μL. It was empirically determined that 2 ng/μL was optimal, allowing for effective allele discrimination for each assay.
The final volume in each assay reaction was 5 μL, composed of 2.5 μL TaqMan GT mastermix (2×) (Applied Biosystems), 0.25 μL assay mix (20×), 2.25 μL DNA (at 2 ng/μL, that is, total input of 4.5-ng template). Each assay included at least two no-template controls and two positive controls. Thermal cycling was performed using the ABI 7900HT instrument (Applied Biosystems) and the following conditions: 95°C, 10 minutes; (95°C, 15 seconds; 60°C, 1 minute) × 40 cycles. If fluorescence values dictated after this cycling, a second cycling of 10× (95°C, 15 seconds; 60°C, 1 minute) cycles and subsequent postread analysis were performed. Sanger sequencing was used to validate all candidate variants.
The final volume in each assay reaction was 5 μL, composed of 2.5 μL TaqMan GT mastermix (2×) (Applied Biosystems), 0.25 μL assay mix (20×), 2.25 μL DNA (at 2 ng/μL, that is, total input of 4.5-ng template). Each assay included at least two no-template controls and two positive controls. Thermal cycling was performed using the ABI 7900HT instrument (Applied Biosystems) and the following conditions: 95°C, 10 minutes; (95°C, 15 seconds; 60°C, 1 minute) × 40 cycles. If fluorescence values dictated after this cycling, a second cycling of 10× (95°C, 15 seconds; 60°C, 1 minute) cycles and subsequent postread analysis were performed. Sanger sequencing was used to validate all candidate variants.
9
Real-time PCR Gene Expression Analysis
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Real-time PCR was conducted using the ABI 7900 HT instrument (Applied Biosystems) following supplier instructions. Taqman® Gene Expression Assay Kits (Applied Biosystems) were used to analyse the gene expression of the PKC gamma coding gene. A panel of nine housekeeping genes were evaluated using excel Normfinder and the four most stable genes were used for normalisation of each experiment.
10
Quantitative Real-Time PCR for Homogeneity and Stability Assessment
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Quantitative real-time PCR (qPCR) measurements to assess the homogeneity and stability were performed by the Institute for Reference Materials and Measurements, Geel, Belgium, using the ABI 7900 HT instrument (Applied Biosystems, Lennik, Belgium). The PCR conditions were the same as those used for the digital PCR measurements. For each concentration level, several vials were selected for the homogeneity and stability studies using a random stratified-sampling approach scheme for the whole batch. Each vial was measured three times in separate qPCR runs, and every measurement result was the mean result from three PCR wells (triplicate). A calibration curve with common plasmid solutions of pIRMM0099, produced independently from the stock solutions of ERM-AD623, was included in every qPCR run. This study design avoided 'between-run' effects by using a common calibrant for the calibration curves on each plate. The qPCR results of the BCR–ABL1 PCR target showed the best method repeatability, so these results were used to assess the homogeneity and stability. The set-up of these experiments is described in detail in the certification report of ERM-AD623.19
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