For the RT-PCR, the TaqMan SARS-CoV-2 Assay Kit version 2 (Thermo Fisher; catalog number CCU002NR) was used, as described previously.7 (link) Each reaction contained 6.25 μL TaqPath 1-Step Multiplex Master Mix, NO ROX (Thermo Fisher; catalog number A28523), 1.25 μL TaqMan SARS-CoV-2 Assay Kit version 2 (primers and probes), 1.00 μL TaqMan MS2 Phage, 11.50 μL nuclease-free water, and 5 μL of sample RNA, nontemplate nuclease-free water control, or TaqMan SARS-CoV-2 Control Kitv2 (CCU002NR) positive control. The one-step RT-PCR was executed on a QuantStudio 5 DX real-time PCR-System (Thermo Fisher; catalog number A36324) machine with the following steps: Uracil N-glycosylase incubation (25°C, 2 minutes), reverse transcription (53°C, 10 minutes), activation (95°C, 2 minutes), and 45 cycles of denaturation (95°C, 3 seconds) and annealing/extension (60°C, 30 seconds).
Quantstudio 5 dx real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio™ 5 Dx Real-Time PCR System is a high-performance real-time PCR instrument designed for diagnostic and research applications. It is capable of performing quantitative PCR analysis with accuracy and precision.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Taqman sars cov 2 assay kit version 2, by Thermo Fisher Scientific (1 mentions)
Taqpath 1 step multiplex master mix no rox, by Thermo Fisher Scientific (1 mentions)
Genup total rna kit, by Biotechrabbit (1 mentions)
Microrna purification kit, by Norgen Biotek (1 mentions)
Oligo d t 18 primers, by Takara Bio (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Takara Bio (1 mentions)
Superscript 3 platinum sybr green, by Thermo Fisher Scientific (1 mentions)
Deparaffinization solution, by Qiagen (1 mentions)
Taqpath 1 step rt qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Mirneasy ffpe kit, by Qiagen (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Nebnext ultra directional rna library prep kit, by New England Biolabs (1 mentions)
Hiseq 1000, by Illumina (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using quantstudio 5 dx real time pcr system
1
SARS-CoV-2 RT-PCR Assay Protocol
2
RNA Isolation and qRT-PCR Analysis
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RNA isolation was performed by GenUP™ Total RNA Kit (Biotechrabbit, Berlin, Germany; Cat #BR0700903) or microRNA Purification Kit (Norgen Biotek, ON, Canada; Cat #21300). Reverse transcription was achieved by RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific™; Cat #K1622, Carlsbad, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems QuantStudio 5 Dx Real-Time PCR System (Thermo Scientific™) with CAPITALTM qPCR Green Mix HRox (biotechrabbit; catalog #BR0501902). Data were analyzed using the 2−ΔΔCt method and normalized to RPL19 expression. All primers used for qRT-PCR can be found in Supplementary Table S1 .
3
Quantitative Analysis of Gene Expression
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Total RNA was isolated from colon tissues using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). First-strand complementary DNA (cDNA) was synthesized from approximately 2 µg of total RNA with Moloney murine leukemia virus reverse transcriptase (Takara, Kyoto, Japan) and Oligo(dT)18 primers (Takara). qRT-PCR was performed using SuperScript™ III Platinum™ SYBR™ Green (Thermo Fisher Scientific) on a QuantStudio™ 5 Dx Real-Time PCR System. The temperature protocol was: 95 °C for 5 minutes, followed by 36 cycles of 95 °C for 15 seconds and 58 °C for 15 seconds, with a final 5-minute extension at 72 °C. The primer sequences used in the study are shown in Table 1 . qRT-PCR analysis was carried out at least 3 times. Relative RNA level was calculated using the 2-ΔΔCT method.
4
Quantitative PCR Workflow for Prostate Cancer
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As with the 46-gene test [Citation19], tissue from FFPE prostate tissue sections with at least 0.5 mm of linear tumor were macro-dissected as directed by the pathologist. Using the miRNeasy FFPE kit and deparaffinization solution (QIAGEN), RNA was extracted from one to five unstained FFPE tissue sections. Due to low RNA concentration after extraction, a preamplification was performed after reverse transcription using TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific, Darmstadt, Germany) for 14 cycles in a single multiplex reaction. Reverse transcription and preamplification were run in triplicate for sample RNA and as single measurements for positive and negative controls. RNA expression levels were then assessed by quantitative PCR in singleplex reactions using a QuantStudio™ 5 Dx Real-Time PCR System (Thermo Fisher Scientific) and a 96-well PCR plate per sample. Each plate also contained positive and negative controls for each gene. The raw quantitative PCR data were analyzed using a web-based tool (Prolaris Biopsy Report Generator; www1.prolaris.com) which checked if the positive and negative controls were within the predefined limits. The web-based tool then calculated the Prolaris Molecular score (CCP score plus four units).
5
IDH1 Expression Analysis in Transduced Cells
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RNA was extracted using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. When required, complementary DNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was used to measure IDH1 expression in control and LV-transduced cells using an IDH1 TaqMan probe (Life Technologies) and the TaqMan Fast Universal PCR Master-Mix (Applied Biosystems). The reaction was performed using the QuantStudio™ 5 Dx Real-Time PCR System (Applied Biosystems) with GAPDH serving as an endogenous control (Life Technologies). Each target gene’s expression was evaluated using a relative quantification approach (2 − ΔΔCT method).
6
RNA Extraction and Sequencing Protocol
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TRI Reagent (Sigma-Aldrich) was used to extract total RNA in compliance with the reagent protocol. qRT-PCR was conducted to measure the RNA level using a QuantStudio 5 Dx Real-Time PCR System (Applied Biosystems, Life Technologies). The oligonucleotides used for qRT-PCR are presented in Table S3 .
For RNA-seq, NEBNext poly(A) mRNA Magnetic Isolation Module was employed to purify the poly(A) mRNA. Then, library preparation was conducted using a NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA). Illumina HiSeq 1000 with paired-end 150-bp read length was used for sequencing. mRNA clean reads were mapped to UCSC hg38 primary assembly genome using TopHat2 software. HTSeq was used to calculate counts among transcripts. Bioconductor package DESeq2 was used for read normalization, size factor estimation, and differential expression analysis, and then bioinformatics analysis was conducted in R expression quantification.
For RNA-seq, NEBNext poly(A) mRNA Magnetic Isolation Module was employed to purify the poly(A) mRNA. Then, library preparation was conducted using a NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA). Illumina HiSeq 1000 with paired-end 150-bp read length was used for sequencing. mRNA clean reads were mapped to UCSC hg38 primary assembly genome using TopHat2 software. HTSeq was used to calculate counts among transcripts. Bioconductor package DESeq2 was used for read normalization, size factor estimation, and differential expression analysis, and then bioinformatics analysis was conducted in R expression quantification.
7
RT-qPCR Validation of miRNA Candidates
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To confirm the findings obtained from PCR array screening, RT-qPCR analysis was performed with a TaqMan miRNA assay as a validation set. For the validation of miRNA candidates, tissue miRNA samples, originating from PDAC patients, were tested by qRT-PCR. cDNA synthesis was performed with TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, CA, USA), according to the manufacturer’s manual. The expression levels of miRNA candidates were detected using TaqMan Universal PCR Master Mix (2X) (Applied Biosystems, CA, USA) under the control of the ViiA 7 real-time PCR system and QuantStudio 5 Dx Real-Time PCR System (Applied Biosystems, CA, USA). Comparative real-time PCR (RT-PCR) was performed in triplicate, including no template controls, and relative expression was calculated using the comparative cross-threshold (Ct) method. Cycle threshold (Ct) value was calculated using ViiA™ 7 Software v1.2 (Applied Biosystems) and QuantStudio 5 Dx (Applied Biosystems) with the threshold set to 0.2. Subsequently, for the normalization of target gene expression level, ΔCt was derived by the following formula: Ct of target gene—Ct of reference gene such as U6 snRNA (endogenous miRNA reference). ΔΔCt was calculated by the following formula: ΔCt of interest group—ΔCt of control group. Then, 2−ΔΔCt was derived as a fold change (FC) of target gene expression.
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