The PCR reactions contained total RNA (100 ng), 5 µl of 2X SYBR green qPCR master mix, 0.08 µl of DNA polymerase, 1 µl of gene specific primer (Table S1) and distilled water up to 10 µl final volume per reaction. Real time PCR reactions were performed in duplicate using the Applied Biosystems QuantStudio 5 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). The 2 -ΔΔCt method was employed for statistical data analysis.
Applied biosystems quantstudio 5 real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems QuantStudio 5 Real-Time PCR System is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It is capable of performing quantitative gene expression studies, genotyping, and other real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Sybr green qpcr master mix, by Vazyme (1 mentions)
Quantifiler trio dna quantification kit, by Thermo Fisher Scientific (1 mentions)
Sybr green reagent, by Yeasen (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
M mlv, by Thermo Fisher Scientific (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Du 730 life science uv vis spectrophotometer, by Beckman Coulter (1 mentions)
Oligo d t 16, by Thermo Fisher Scientific (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
24 protocols using applied biosystems quantstudio 5 real time pcr system
1
Quantitative Real-Time PCR Assay
2
SYBR Green qPCR Gene Expression Protocol
3
RNA Isolation, Reverse Transcription, and RT-qPCR Analysis
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Total RNA was isolated from the tissues or cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. Reverse transcription was performed with ReverTra Ace® qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's protocol (20 µl reaction: 14 µl RNA+ 4 µl 5xRT Buffer+ 1 µl Primer Mix+ 1 µl EnzymeMix, step 1 16°C for 5 min, step 2 42°C for 30 min, step 3 98°C for 5 min). The RT-qPCR analysis was performed using the Applied Biosystems QuantStudio 5 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with THUNDERBIRD SYBR qPCR mix (Toyobo Co., Ltd.) according to the manufacturer's protocol (20 µl reaction: 1 µl cDNA+ 7 µl RNA-free water+ 0.8 µl forward primer+ 0.8 µl reverse primer+ 0.4 µl ROX+ 10 µl THUNDERBIRD SYBR qPCR mix, the concentration of primer was 0.2 µM, step 1 95°C for 2 min, step 2 95°C for 15 sec, step 3 60°C for 60 sec, repeat step 2 and step 3 for 40 cycles, final step 72°C for 5 min). GAPDH was used as internal control. The relative expression levels were calculated using the 2−ΔΔCq equation (20 (link)). The primer sequences for PCR were as follows: CITED1, forward 5′-AGGATGCCAACCAAGAGATG-3′ and reverse 5′-GTTTAGTGGGAGGGGTGGTT-3′; GAPDH, forward 5′-GGTCGGAGTCAACGGATTTG-3′ and reverse 5′-ATGAGCCCCAGCCTTCTCCAT-3′.
4
Circular RNA hsa_circ_0126897 in Colorectal Cancer
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The tumor tissues and paracancerous specimens from four CRC patients were collected, after the patients had signed informed consent. The target gene of hsa_circ_0126897_CBC1 was found to be SLC4A4 from circBase (http://circrna.org/cgi-bin/singlerecord.cgi?id=hsa_circ_0126897 ). The SYBR® Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd., Shiga, Japan) and the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Foster City, CA, USA) were used to detect the mRNA levels of hsa_circ_0126897_CBC1 and SLC4A4 according to the manufacturer's instructions. All the primers were designed and synthesized by Takara Biotechnology Co., Ltd. (Beijing, China). The primers of hsa_circ_0126897_CBC1: 3′-CTCATGGTGATGCTGAACCTTCTTA-5′ and 3′-CTCATGGTGATGCTGAACCTTCTTA-5′ (139 bp); and SLC4A4: 3′-AGCACTCTATACGCCCCAAG-5′ and 3′-TTCCTTTTCTCTCACGCCCT-5′ (540 bp). In addition, β-actin was used as a reference, and the primer sequences were 5′-CTACAATGAGCTGCGTGTGG-3′ and 5′-AGGCATACAGGGACAACACA-3′ (308 bp).
5
Profiling of iCCA Tumor Transcriptome
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Tissue samples were collected as pairs, i.e., tumor tissue and adjacent normal tissue, from 10 patients with iCCA undergoing surgery at Peking Union Medical College Hospital. A total of 6 male and 4 female patients with mean age of 62 (range, 54–68) years were included. The collected tissue samples were stored in a refrigerator at −80°C. All patients were enrolled from November 2018 to April 2019. The study was approved by the Clinical Research Ethics Committee of Peking Union Medical College Hospital. Each patient provided a written informed signed consent. Total RNA was isolated from each sample with Trizol LS reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and then used for cDNA synthesis using oligo(dT)primers and SuperScript™ III Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). PCR Master Mix (2X) (Superarray) and Applied Biosystems QuantStudio5 Real-time PCR system (Thermo Fisher Scientific, Inc.) were utilized for RT-qPCR. The sequences of primers for selected hub genes and housekeeping gene (β-actin) are shown in Table SI .
6
qPCR Analysis of Circadian Transcripts
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RNA was isolated using Qiagen RNeasy Micro Kit, as per manufacturer's instructions, and quantified using Nanodrop 8000 (Thermo Fisher Scientific, Waltham, MA, USA). The isolated RNA was converted to cDNA using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), as per manufacturer's protocol. The template cDNA was amplified using Qiagen Primers for the transcripts: Hsf1 (NM_008296), Rbm3 (NM_016809), Cirbp (NM_007705) and Prok2 (NM_015768) to quantify levels of mRNA in the SCN and LHb. GAPDH (NM_008804) was used as the reference housekeeping gene in both SCN and LHb, as reported previously [34 (link)–39 (link)]. Non-template controls were run for each primer pair. qPCR was carried out using Applied Biosystems QuantStudio5 Real-Time PCR System (Thermo Fisher Scientific). All experimental groups for qPCR experiments consisted of four biological replicates split into three technical replicates to ensure accuracy of measurement. The average of the threshold cycle (Ct) was taken across the triplicates and normalized to the GAPDH using the 2(ΔCt) method. Fold changes were calculated relative to controls using the 2(ΔΔCt) method.
7
Quantitative Viral RNA and Gene Expression Analysis
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The viral RNA copies in the samples collected from the cells and animals were determined as described previously [63 (link)]. Total RNA was isolated with TRIzol reagent (Invitrogen), 1 μg of total RNA was used for reverse transcription (Vazyme). qPCR was performed on the Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo Fisher) with SYBR green qPCR Master Mix (Vazyme) according to the manufacturer’s instructions. The ACE2 mRNA copies were calculated based on a calibration curve obtained by 10-fold stepwise dilution of plasmid DNA. To determine the relative levels of viral RNA (the viral primers valid to detect the genomic RNA or subgenomic RNA) or CACNA1C mRNA in cells, the 2-ΔΔCT method was used [64 (link)]. The cycle threshold (CT) of each transcript minus the corresponding CT of the internal control gene 28S rRNA or β-actin was calculated as:
ΔCT sample means ΔCT experimental sample 1, ΔCT experimental sample 2, ΔCT experimental sample 3, ΔCT control sample 1, ΔCT control sample 2, or ΔCT control sample 3.
The relative expression of viral RNA or CACNA1C mRNA relative to control samples was calculated as:
Experiments were done in three biological replicates. The primer sequences of individual genes are listed inS1 Table .
ΔCT sample means ΔCT experimental sample 1, ΔCT experimental sample 2, ΔCT experimental sample 3, ΔCT control sample 1, ΔCT control sample 2, or ΔCT control sample 3.
The relative expression of viral RNA or CACNA1C mRNA relative to control samples was calculated as:
Experiments were done in three biological replicates. The primer sequences of individual genes are listed in
8
DNA Profiling from Bloodstain Samples
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Bloodstain samples from mutational father–son pairs were extracted using the magnetic beads on the Microlab STAR Liquid Handling System (Hamilton, Bonaduz, Switzerland). Genomic DNA was quantified on the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific) using the Quantifiler® Trio DNA Quantification Kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations [23 ]. Library preparation was performed using the ForenSeq™ DNA Signature Prep Kit (Verogen, San Diego, CA, USA) according to the manufacturer’s recommendations [24 ]. Hereof, one nanogram purified DNA (5 μL input volume at a concentration of 0.2 ng/μL) was amplified using DNA Primer Mix A. MPS was performed on the MiSeq FGx® Forensic Genomics System (Verogen) using the MiSeq FGx® Reagent Kit following the manufacturer’s instruction [25 ] with two modifications: (i) 52 libraries were pooled for a run, including 50 samples, a positive control, and a negative control; (ii) 7 μL pooled libraries was added into 591 μL Hybridization Buffer (HT1) and then mixed with 4 μL Human Sequencing Control (HSC) mixture, not 2 μL HSC in the previous study [20 (link)]. MPS raw data were processed by the ForenSeq™ Universal Analysis Software (UAS) v1.3 (Verogen) at default analysis thresholds [26 ].
9
Quantitative Gene Expression Analysis
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Total RNA was extracted from the cell pellet using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Takara, Shiga, Japan) according to the manufacturer’s protocol. Quantitative PCR was performed on the Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, IL, USA) using SYBR Green reagents (Yeasen Biotech, Shanghai, China); the corresponding primers are listed in Supplementary Table 2 . The expression of target genes was calculated using the 2−ΔΔt method after normalizing to the housekeeping gene β-actin and control group.
10
Quantification of Sirpa and Actb Expression
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RAW264.7 cells were either stimulated with 100 ng/mL LPS or infected with L. donovani (MOI: 50) for 24 h as described above. RNA was extracted using a TRIzol reagent (Invitrogen). The concentration of total RNA was measured using a DU730 Life Science UV/vis spectrophotometer (Beckman Coulter, Chaska, MN, USA), and 4 µg of total RNA was used as the template for the synthesis of cDNA. A tube containing 500 ng of oligo (dT)16 and 10 nmol of dNTPs (Fisher Scientific, Loughborough, UK) with template RNA was incubated for 5 min at 65 °C. Then, 5× first-strand buffer, 200 nmol of DTT (Thermo, Waltham, MA, USA), and 200 U of M-MLV (Thermo) were added, and the tube was incubated at 37 °C for 50 min. The reaction was stopped by incubation for 15 min at 70 °C. The synthesized cDNA was used for the expression analyses of Sirpa and Actb. The designed primers are listed in Table 1 [15 (link)]. A real-time polymerase chain reaction (PCR) assay was conducted using 1 µL of reverse transcription PCR product as the template and 10 µL of SYBR Select Master Mix (Thermo) with the Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo). Data were analyzed with 2−ΔΔCt methods through normalization with Actb. The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
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