mRNA extractions from cultured cells were performed with the Qiagen miRNeasy kit (217004). Reverse transcription of total RNA (500 ng) into cDNA was performed with PrimeScript (Takara, RR036Q). All qRT-PCR reactions were performed as triplicates in 7900HT qPCR instrument (Applied Biosystems). Results were normalized to the housekeeping genes beta-actin or GAPDH (for human) and Rpl19 or GAPDH for mouse. For western blotting, cultured cells were lysed in RIPA lysis buffer (Thermo Fisher Scientific, 89900), and protein concentrations were determined using Bradford 1X dye reagent (Bio-Rad). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking the membranes with 5% BSA in TBST buffer for 1 hr at room temperature, primary antibodies were incubated at 4°C overnight, and then secondary antibodies were incubated for 1 hr at room temperature. Finally, the membranes were visualized with an enhanced chemiluminescence reagent (Thermo Fisher Scientific).
7900ht qpcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7900HT qPCR instrument is a real-time PCR system designed for quantitative gene expression analysis. It provides precise and sensitive detection of target sequences in a variety of sample types. The instrument utilizes thermal cycling, fluorescence detection, and advanced data analysis software to enable accurate and reproducible quantification of nucleic acids.
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Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Bradford 1x dye reagent, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Qscript xlt cdna supermix, by Quantabio (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taqman primer, by Thermo Fisher Scientific (1 mentions)
Tissuelyzer 2, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
High capacity cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Gotaq probe qpcr master mix, by Promega (1 mentions)
Nanodrop instrument, by Thermo Fisher Scientific (1 mentions)
Superscript first strand kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Sypro orange dye, by Thermo Fisher Scientific (1 mentions)
9 protocols using 7900ht qpcr instrument
1
RNA Extraction and RT-qPCR Analysis
2
Quantifying Gene Expression in Cecal Epithelium
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Intact ceca or enriched cecal epithelial cells were collected in Qiazol (Qiagen), and homogenized using a Tissue Lyzer II (Qiagen). Total RNA was extracted using the RNA Microarray Kit (Qiagen) and quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized from total RNA using the High Capacity cDNA Synthesis Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Quantitative PCR was carried out using Taqman primers (Thermo Fisher Scientific) on a 7900HT qPCR instrument (Applied Biosystems/Thermo Fisher Scientific). The mRNA expression of each gene tested were normalized to naïve WT samples and assessed using relative quantification method. GAPDH was used as an endogenous control.
3
Optimizing qPCR Assays for GM Alfalfa Detection
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A 7900 H T qPCR instrument (Applied Biosystems, Foster City, CA, USA) was applied for determination of optimal oligo concentrations for the newly developed event-specific gm alfalfa qPCR methods. PCR primer concentrations varied between 200 nM and 500 nM, whereas PCR probes concentrations varied between 100 nM and 300 nM. The qPCR assays were performed in a 25 μL reaction volume containing 1× GoTaq Probe qPCR Master Mix (Promega GmbH, Mannheim, Germany), oligos in different concentrations, 5 μL plasmid and nuclease-free water ad 25 μL. Plasmids were diluted 1:105 (pJ101 ≅ 2.54 × 106 copies) or 1:106 (pJ163 ≅ 2.88 × 105 copies; pKK179 ≅ 1.5 × 105 copies) and analyzed in six replicates for each oligo concentration and combination. Cycling conditions were the following: initial denaturation for 3 min at 95 °C and 45 cycles of 95 °C for 15 s and 60 °C for 60 s. All following qPCR experiments described in this work were performed using the optimized oligo concentrations (see Table 2 ) and cycling conditions (except for robustness tests; see robustness test section). Optimal concentrations were selected based on PCR efficiency, amplification curves and Cq values.
4
Quantitative RT-PCR Analysis of BM-MSCs
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An eppendorf (EP) tube and tip (free of RNase enzymes) were used. Total ribonucleic acid (RNA) was extracted from the BM-MSCs or tissues. The quantity and quality of total RNA were assessed using a nanodrop instrument (Thermo Fisher Scientific) and 50–100 ng of RNA was reverse-transcribed from random hexamers with Superscript first strand kit (Invitrogen, Carlsbad, CA, USA). QRT-PCR was performed using a 7900HT qPCR instrument according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The primer sequences are shown in Table 1 . The measurement was repeated three times.
5
Quantification of miRNA-199a levels
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Total RNA was isolated from the cells or EVs using Trizol reagent (Invitrogen, USA) with standard conditions. RNA was converted to cDNA using random primers and SuperScript II reverse transcriptase enzyme or using TaqMan microRNA assays (Invitrogen, USA) as previously described [52 (link)]. qRT-PCR was performed in an Applied Biosystems 7900HT qPCR instrument. A custom TaqMan probe (LNA sequence TG+AGCC+T+GG+GA) was designed to the modified pre-miR-199a loop amplified using forward primer 5′- CCCAGTGTTCAGACTACCTGTTC-3′ and reverse primer 5′- TAACCAATGTGCAGACTACTGT-3′. Data were normalised to U6 or 18S rRNA and the relative expression was determined using the 2−ΔCT method.
6
Quantitative RT-PCR for Epo Transcripts
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For indicated conditions and time-points, tissues were collected and minced followed by RNA extraction using TRIzol Plus RNA Purification Kit (Invitrogen Cat#: 12183555). cDNA synthesis was performed using qScript XLT cDNA SuperMix (QuantaBio Cat# 95048-025). After reverse transcription, qPCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) and analyzed using an Applied Biosystems 7900 HT qPCR instrument. The cycle threshold (Ct) value for each transcript was normalized to Gapdh. The comparative Ct method was used to quantify transcript abundance. TaqMan Assays used were; Epo: Mm01202755_m1, Gapdh: Mm99999915_g1 Gapdh.
7
Quantitative Transcriptome Analysis in C. elegans
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For indicated conditions and time-points, worms were washed off the plate with M9 buffer followed by three further washes to remove external bacteria, and rocked for 0.5 hr in M9 buffer to clear bacteria from the gut. RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. cDNA synthesis was performed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) or qScript XLT cDNA SuperMix (QuantaBio). After reverse transcription, qPCR was performed using TaqMan Gene Expression Assays (Applied Biosystems) and analyzed using an Applied Biosystems 7900 HT qPCR instrument. The cycle threshold (Ct) value for each transcript was normalized to either act-1, act-2b or nhr-23. For pathogen experiments, all values were normalized using control genes nhr-23 or tba-1. The comparative Ct method was used to quantify transcript abundance. At least three biological replicates with duplicate technical replicates were used for qPCR experiments. TaqMan Assays are listed in Key Resources Table.
8
RNA Extraction and RT-qPCR Analysis
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mRNA extractions from cultured cells were performed with the Qiagen miRNeasy kit (217004). Reverse transcription of total RNA (500 ng) into cDNA was performed with PrimeScript (Takara, RR036Q). All qRT-PCR reactions were performed as triplicates in 7900HT qPCR instrument (Applied Biosystems). Results were normalized to the housekeeping genes beta-actin or GAPDH (for human) and Rpl19 or GAPDH for mouse. For western blotting, cultured cells were lysed in RIPA lysis buffer (Thermo Fisher Scientific, 89900), and protein concentrations were determined using Bradford 1X dye reagent (Bio-Rad). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking the membranes with 5% BSA in TBST buffer for 1 hr at room temperature, primary antibodies were incubated at 4°C overnight, and then secondary antibodies were incubated for 1 hr at room temperature. Finally, the membranes were visualized with an enhanced chemiluminescence reagent (Thermo Fisher Scientific).
9
Thermal Shift Assay of gp63.1ht Variants
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A SYPRO Orange-based thermal shift assay (Lo et al., 2004) was performed in a 384-well plate with the help of a 7900HT qPCR instrument (Applied Biosystems, Foster City, CA) (Lavinder et al., 2009) , on the following gp63.1ht variants: wild-type, D470A, H473A. All proteins had a concentration of 0.5 mg/ml. The stock solution of the SYPRO Orange dye (Thermo Fisher Scientific) was diluted 5,000 times to yield a nominal concentration (1 X dilution). Two negative controls that either did not contain the protein or both the protein and the dye were tested. Both controls showed no change in fluorescent signal associated with protein unfolding. All statistical analyses were conducted in R 3.0.2. For the purpose of visualization, fluorescence intensity was normalized within each well to values between 0 and 1 using the formula: F norm 5 (F raw -F min )/ (F max -F min ). The normalized fluorescences' were then averaged, smoothed and plotted against the temperature. The melting temperatures were defined as the inflexion points of a Boltzmann sigmoidal curve fitted using a nonlinear least-square regression on the raw fluorescence data.
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