Complementary DNA (cDNA) was synthesized directly from treated and untreated human WBC using FastLane Cell cDNA Kit (Qiagen, Hilden, Germany). The purity was checked and quantified using the NanoDrop spectrophotometer ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA). Quantitative real-time PCR was performed with the SYBR Green PCR Master Mix (Qiagen, Chatsworth, CA, USA) in an ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. The mRNA levels of IL-4, IL-10, IL-12B, IL-16, and the reference gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were assayed using gene-specific SYBR Green-based QuantiTect® Primer Assays (Qiagen, Hilden, Germany) according to manufacturer’s instructions. A 25-μL reaction volume was used in each well of the PCR plates. Briefly, 12.5 μL of master mix, 2 μL of assay primer and 2 μL of template cDNA (500 pg) were added to each well. Reaction mixtures were incubated for initial denaturation at 95 °C for 5 min, followed by 40 PCR cycles. Each cycle consisted of 95 °C for 5 s and 60 °C for 30 s. The relative quantification of the aforementioned gene expression was analyzed using an ABI 7500 fast real-time PCR system (Applied Biosystems). The values were expressed as fold changes over the control and expressed as means with their standard errors.
Abi 7500 fast real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Japan, Germany, United Kingdom, Switzerland, Canada, Lithuania, Austria
The ABI 7500 Fast Real-Time PCR System is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It is capable of performing fast and precise DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (334 mentions)
Primescript rt reagent kit, by Takara Bio (121 mentions)
Trizol, by Thermo Fisher Scientific (91 mentions)
Rneasy mini kit, by Qiagen (88 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (74 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (54 mentions)
Sybr premix ex taq, by Takara Bio (50 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (42 mentions)
Iscript cdna synthesis kit, by Bio-Rad (33 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (33 mentions)
Trizol reagent, by Takara Bio (29 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (28 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (27 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (27 mentions)
Sybr green pcr kit, by Takara Bio (25 mentions)
Rnaiso plus, by Takara Bio (23 mentions)
M mlv reverse transcriptase, by Promega (23 mentions)
Itaq universal sybr green supermix, by Bio-Rad (20 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (19 mentions)
Sybr premix ex taq 2, by Takara Bio (18 mentions)
1 243 protocols using abi 7500 fast real time pcr system
1
Real-Time PCR Analysis of Cytokine Expression in Human WBC
2
Total RNA Isolation and qRT-PCR Analysis
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Total RNA isolation and cDNA was synthesized as explained earlier [56 (link),57 (link)]. Total RNA from different organs/tissues and flower developmental stages was extracted using a HiPure plant RNA mini kit (Magen, Guangzhou, China) according to the manufacturer’s suggestions. In total RNA, genomic DNA contamination was removed by DNase I. The qRT-PCR analysis was executed in an ABI 7500 fast real-time PCR system (Applied Biosystems, MA, USA) using iTaqTM Universal SYBR Green Supermix (BIO-RAD, CA, USA) following the manufacturer’s protocols. PCR was performed in a total volume of 20 µL containing 10 µL iTaq ™ Universal SYBR Green Supermix (BIO-RAD), 7.2 µL of ddH2O, 0.4 µL each of forward and reverse primers, and 2 µL of cDNA, using an ABI 7500 fast real-time PCR system (Applied Biosystems, USA). GAPDH was used for normalization of data and the 2 °C-ΔΔCT method was employed for measuring the relative expression analysis [58 (link)]. The reactions were performed in triplicate.
3
Quantifying miRNA-6716-5p Expression in Colorectal Cancer
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RT-qPCR was performed on the ABI 7500 Fast Real-Time PCR systems (Applied Biosystems, Foster City, CA, USA). Total RNA and miRNA were extracted from CRC cells and tissues using MiPure Cell/Tissue miRNA Kit (Vazyme, Nanjing, People’s Republic of China). cDNA was synthesized by Universal cDNA Synthesis Kit (Roche, USA). The primer mix contained a specific stem-loop primer of miR-6716-5p and a RNU6A (U6) reverse primer for cDNA synthesis of miRNA. Random hexamer primer was used to synthesize the cDNA of the mRNAs. RT-qPCR was performed using the FastStart Universal SYBR Green Master (Roche) in the ABI 7500 Fast Real-Time PCR system (Applied Biosystems). GADPH and U6 were used as endogenous controls. A melting curve analysis was performed for the primer sets, and each showed a single peak indicating the specificity of the primers tested. Relative expression levels were computed using the 2−ΔΔCt method. The primer sequences used are listed in the Supplemental Information.
4
Quantitative Analysis of PIGT Expression
5
Gene Expression Analysis by RT-qPCR
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Total RNA from the cells was prepared using a Trizol reagent according to the manufacturer’s protocol (TaKaRa, China). Reverse transcription of total RNA to cDNA was performed using a first-strand cDNA synthesis kit (Roche Diagnostics, USA). Quantitative real-time PCR analysis was performed using SYBR Select Master Mix (ThermoFisher, USA) and gene-specific primers (Genepharma, China) on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, USA). mRNA expression data were normalized to that of GAPDH. For miRNA detection, total RNA and miRNA were isolated with Trizol reagent using DirectzolTM RNA Miniprep (Zymo Research, CA, USA). RNA was reverse-transcribed using the miScript Reverse Transcription kit (Qiagen, Germany) according to the manufacturer’s instructions. Quantitative PCR was performed using the miScript SYBR Green PCR kit (Qiagen, Germany) and miScript Primer assays (Qiagen, Germany) for miRNAs and RNU6 as an internal control. miRNAs were detected on ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Relative expression values were normalized to the internal control using the 2-∆∆ Ct method. Primer sequences are listed in Supplementary Table 2 .
6
Gene and miRNA Expression Analysis
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For mRNA: Total RNA from tissues and cells was prepared using Trizol reagent according to manufacturer's protocol (Invitrogen, CA, USA). Reverse transcription of total RNA into cDNA was performed using first strand cDNA synthesis kit (Roche Diagnostics, IN, USA). Quantitative Real-Time PCR analysis was performed using SYBR Select Master Mix (Thermofischer Scientific, Waltham, MA, USA) and gene specific primers (Eurofins MWG Operon, Ebersberg, Germany) on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). mRNA expression data were normalized to GAPDH. Primer sequences were listed in Supplementary CTAT.
For miRNA: Total RNA along with miRNA was isolated with Trizol reagent by Directzol TM RNA Miniprep (Zymo Research, Irvine, CA). RNA was reverse transcribed using miScript Reverse Transcription kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. A quantitative PCR was performed using miScript SYBR Green PCR kit (Qiagen, Germany) and miScript Primer assays (Qiagen, Germany) for miR-1224 with RNU6 as an internal control. miRNAs was detected on ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The relative expression values were normalized to the internal control by using 2 -ΔΔCt . Primer sequences were listed in Supplementary CTAT.
For miRNA: Total RNA along with miRNA was isolated with Trizol reagent by Directzol TM RNA Miniprep (Zymo Research, Irvine, CA). RNA was reverse transcribed using miScript Reverse Transcription kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. A quantitative PCR was performed using miScript SYBR Green PCR kit (Qiagen, Germany) and miScript Primer assays (Qiagen, Germany) for miR-1224 with RNU6 as an internal control. miRNAs was detected on ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The relative expression values were normalized to the internal control by using 2 -ΔΔCt . Primer sequences were listed in Supplementary CTAT.
7
Quantifying miRNA and mRNA Expression via qRT-PCR
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For miRNA analysis, the first-strand cDNA was Table 1. List of the qRT-PCR primers used in this study synthesized using the Mir-X™ miRNA First-Strand Synthesis Kit (TaKaRa, Japan). qRT-PCR was performed using SYBR Premix Ex Taq II (TaKaRa, Japan) on an ABI 7500 fast real-time PCR system (Applied Biosystems, USA) and using U6 snRNA as endogenous control.
For mRNA analysis, the first-strand cDNA was synthesized using the Primescript ® RT reagent kit with gDNA Eraser (TaKaRa, Japan). qRT-PCR was performed using SYBR Select Master Mix (Applied Biosystems, USA) on an ABI 7500 fast real-time PCR system (Applied Biosystems, USA) and using beta-actin as endogenous control.
A dissociation curve was performed to ensure the validity of each specific PCR product. The experiments were independently performed three times in triplicate. All samples were normalized to endogenous control and determined using the 2 -ΔΔCt -analysis methods. The primers that were used in the qRT-PCR are listed in Table 1.
For mRNA analysis, the first-strand cDNA was synthesized using the Primescript ® RT reagent kit with gDNA Eraser (TaKaRa, Japan). qRT-PCR was performed using SYBR Select Master Mix (Applied Biosystems, USA) on an ABI 7500 fast real-time PCR system (Applied Biosystems, USA) and using beta-actin as endogenous control.
A dissociation curve was performed to ensure the validity of each specific PCR product. The experiments were independently performed three times in triplicate. All samples were normalized to endogenous control and determined using the 2 -ΔΔCt -analysis methods. The primers that were used in the qRT-PCR are listed in Table 1.
8
Quantifying mtDNA Copy Number via qRT-PCR
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DNA was extracted using a QIAGEN DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer’s protocol. Quantification of mtDNA copy number was performed using qRT-PCR. The ND1 gene fragment of the mitochondrial genome was amplified from all the individuals using the primer ND1-F (5’-CCCTAAAACCCGCCACATCT-3’) and ND1-R (5’-GAGCGATGGTGAGAGCTAAGGT-3’). The APP gene fragment of the nuclear genome was amplified from all the individuals using the primer APP-F (5’-TGTGTGCTCTCCCAGGTCTA-3’) and APP-R (5’-CAGTTCTGGTCACTGG-3’). QRT-PCR was performed with an initial denaturation step of 95°C for 20 sec, then 95°C denaturation for 3 sec, followed by primer and probe hybridization and DNA synthesis at 60°C for 30 sec; the last two steps were repeated for 40 cycles. The reaction was measured using the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, USA).
9
Quantifying NUFIP1 mRNA Expression
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Total RNA was extracted from cell line samples using TRIZOL (Thermo Fisher Scientific; Carlsbad, CA, USA) and converted to cDNA using reverse transcription-PCR (Thermo Fisher Scientific; Carlsbad, CA, USA). The resulting cDNA, or a commercial tissue cDNA array (Shanghai Outdo Biotech; Shanghai, China), was used to measure levels of NUFIP1 mRNA using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the SYBR Premix Ex Tag (Thermo Fisher Scientific; Carlsbad, CA, USA). The following cycling conditions were used: 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Levels were quantified using the comparative Ct method and normalized to levels of GAPDH mRNA. The sequences of primers are listed in Supplementary Table S2
10
Quantifying CISD2 and Beclin1 Knockdown
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After transfection with CISD2 shRNA, Beclin1 shRNA, or the corresponding Ctrl shRNA for 48 h, total RNA was extracted with TRIzol (Invitrogen, Thermo Fisher Scientific, USA). Then, a 1/5 volume of chloroform was added for extraction and centrifugation to obtain the upper clear liquid phase, and the same volume of isopropanol was then added and stored at −20°C overnight. cDNA was obtained by reverse transcription with a PrimeScript™ RT Reagent Kit (Promega, USA) in the Promega GoScript reverse transcription system (A5000). Real-time PCR analysis was performed using Promega GoTaq® qPCR Master Mix in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA). With 18S rRNA as the internal reference, qPCR was carried out in a 20 μl reaction system. The 2−ΔΔCt method was used to analyze the data. Three complex wells were set up for all reactions, and the experiment was repeated three times.
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