RNA was extracted with the Nucleospin II kit (Macherey-Nagel) and reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCRs were performed either using TaqMan assays with qRT-PCR Mastermix Plus without UNG (Eurogentec) or using SYBR green (Applied Biosystems). Oligonucleotides were purchased from MWG Eurofins Genomics (Supplementary Data ). Reactions were run on an ABI/PRISM 7500 instrument and analyzed using the 7500 system SDS software (Applied Biosystems).
Abi prism 7500 instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
The ABI PRISM 7500 is a real-time PCR instrument designed for quantitative gene expression analysis, SNP genotyping, and other real-time PCR applications. It features 96-well thermal cycling, a flexible optical detection system, and software for data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
7500 system sds software, by Thermo Fisher Scientific (1 mentions)
Nucleospin 2 kit, by Macherey-Nagel (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix kit, by Takara Bio (1 mentions)
Taqman gene expression cells to ct kit, by Thermo Fisher Scientific (1 mentions)
Sybr green real time pcr master mix, by Thermo Fisher Scientific (1 mentions)
Qiaamp rna blood kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Nanodrop 1000 detector, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Takara Bio (1 mentions)
Sybr green rt pcr kit, by Takara Bio (1 mentions)
No amperase ung, by Thermo Fisher Scientific (1 mentions)
Specific taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
19 protocols using abi prism 7500 instrument
1
qRT-PCR Transcriptome Analysis Protocol
2
Quantitative RT-PCR for Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from tissues and cells by Trizol reagent (CA, USA). Then, the cDNA was synthesized with reverse transcription Kit (Applied Biosystems, Japan). Quantitative RT-PCR was performed using an ABI Prism 7500 instrument (Applied Biosystems) and SYBR Green PCR Master Mix kit (Takara, Japan). GAPDH was used as internal reference. The relative expression level of related genes was calculated by 2−△△Ct method. All primers are provided by SANGON biotech (China) and are listed in Supplementary Table 1 .
3
Quantitative Analysis of Stress Response Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA isolation (5 µM PS incubation for 2 h, 6 h, 12 h, and 24 h) and cDNA synthesis were performed with the TaqMan Gene Expression Cells-to-CT kit (Ambion, Foster City, CA). Quantitative real-time PCR (qRT-PCR) reactions were conducted using SYBR Green Real-Time PCR Master Mix (Ambion) with 15 µl reaction volume. The qRT-PCR primers were ordered from Oligomer (Helsinki, Finland) using sequences from Kokot et al. [25 (link)] for Nrf2, GSTP1, and HO-1, the reference gene sequence for acid riboprotein P0 (RPLP0) from Malinen et al. [26 (link)], and intron-spanning qPCR primers for human p62 mRNA (forward 5′-GCA CAC CAA GCT CGC ATT C-3′ and reverse 5′-ACC CGA AGT GTC CGT GTT TC-3′) that detect human p62 mRNA isoforms 1–3 (NM_003900.4 ) were designed using the OLIGO program (Molecular Biology Insights, Cascade, CO) and the BLAST algorithm. Samples were analyzed in triplicate in an ABI Prism 7500 instrument (Applied Biosystems, Foster City, CA) with the following reaction conditions: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s, 58 °C for 20 s and 72 °C for 20 s, final annealing 72 °C for 5 min, and redenaturation 95 °C for 1 min. Finally, the specificity of the primers was verified by performing a melt curve analysis. Data were obtained using the comparative Ct method and expressed as fold-change normalized against RPLP0.
4
Quantification of MBD2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was purified using QIAamp® RNA Blood Kit (Qiagen GmbH, Hilden, Germany). An amount of 250 ng of each RNA sample was reverse transcribed to cDNA (QuantiTect Reverse Transcription kit; Qiagen). The cDNAs were further examined using the quantitative polymerase chain reaction (qPCR) Primer Assay for Human MBD2 (Cat. #PPH08621A-200; Qiagen). The real-time PCR (RT-PCR) amplification was performed on an ABI PRISM® 7500 instrument and the results were analyzed with ABI PRISM® Sequence Detection Software, ver.1.4.0 (Applied Biosystems, Foster City, CA, USA). The total volume of each reaction was 50 µL with less than 100 ng cDNA per reaction. The amplification conditions of each RT-PCR cycle were as follows: denaturation at 94 °C for 15 seconds, primer annealing at 55 °C for 30 seconds, followed by primer extension at 72 °C for 30 seconds. The expression levels of MBD2 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the ∆∆CT method. In order to avoid contamination, we included an internal non template control in each run.
5
Genotyping of Inflammation-Related SNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from blood samples at the Laboratory Medical Oncology (VUmc, Amsterdam, The Netherlands) using the QIAamp DNA Mini-Kit according to the manufacturer protocol (Qiagen, San Diego, CA). The concentration and purity of DNAs was determined with the NanoDrop-1000-Detector (NanoDrop-Technologies, Wilmington, USA). Genotype analysis of rs1800796, rs6136, and rs1130233 polymorphisms was performed using Taqman-based PCR reactions carried out in 12.5 µl total volume, using 20 ng of DNA diluted in TaqMan Universal Master Mix with specific primers and probes (SNP Genotyping Assays products C__11326893_10, C__11975277_20, and C__7489835_10, respectively). The ABIPRISM-7500 instrument (Applied Biosystems, Life Technologies, Foster City, CA) equipped with the SDS version-2.0 software was employed to evaluate the allelic content of each sample in the plate by reading the generated fluorescence.
6
Quantifying PD-L1 Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted with Trizol (Takara, Japan), and the cDNA was reverse-transcribed from the total RNA by an RNA reverse transcription kit (Applied Biosystems, USA). An SYBR Green RT-PCR kit (Takara, Japan) and an ABI prism 7500 instrument (Applied Biosystems, USA) were used for RT-PCR, and the reaction conditions were as follows: pre-denaturation at 95 °C for 3 min, 40 cycles (95 °C 30 s, 60 °C 45 s), and extension at 72 °C for 6 min. The primers were synthesized by Shanghai Sangong Biotechnology Co., Ltd. (China), and they are summarized below:
PD-L1 forward: 5′- TGCGGACTACAAGCGAATCA-3′; PD-L1 reverse: 5′- GATCCACGGAAATTCTCTGGTT-3′; GAPDH (internal control) forward: 5′-CCAGGTGGTCTCCTCTGA-3′; GAPDH (internal control) reverse: 5′-GCTGTAGCCAAATCGTTGT-3′. The relative gene expression levels were calculated by the 2-△△Ct method.
PD-L1 forward: 5′- TGCGGACTACAAGCGAATCA-3′; PD-L1 reverse: 5′- GATCCACGGAAATTCTCTGGTT-3′; GAPDH (internal control) forward: 5′-CCAGGTGGTCTCCTCTGA-3′; GAPDH (internal control) reverse: 5′-GCTGTAGCCAAATCGTTGT-3′. The relative gene expression levels were calculated by the 2-△△Ct method.
7
Mitochondrial Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mRNA expression of selected genes was determined in detail elsewhere.32 (link)
In brief, the determination of the mRNA expression of genes of interest MT-ND5 (gene encoding ND-5 subunit of mitochondrial enzyme complex-1, ie, NADH dehydrogenase 5); NDUFA 12 (gene encoding subunit 12 of NADH:ubiquinone oxidoreductase), SDHA (gene encoding flavoprotein subunit A of the complex of succinate dehydrogenase), CYC1 (gene encoding cytochrome c1), COX4I1 (gene encoding COX4I1 subunit of cytochrome c oxidase), ATP50 (gene encoding ATP50 subunit of ATP synthase), DLAT (gene encoding dihydrolipoamide-S-acetyltransferase, E2 subunit of PDH) and CS (gene encoding citrate synthase) was performed using an ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA, USA) with TaqMan® Universal PCR Master Mix, NO AmpErase® UNG and specific TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). PCR amplifications were performed at least in duplicate in a total reaction volume of 20 μL. The expression of beta-2-microglobulin (B2M) was used to compensate for variations in input RNA amounts and the efficiency of reverse transcription. The modified formula 2−∆Ct was used to calculate relative gene expression.
In brief, the determination of the mRNA expression of genes of interest MT-ND5 (gene encoding ND-5 subunit of mitochondrial enzyme complex-1, ie, NADH dehydrogenase 5); NDUFA 12 (gene encoding subunit 12 of NADH:ubiquinone oxidoreductase), SDHA (gene encoding flavoprotein subunit A of the complex of succinate dehydrogenase), CYC1 (gene encoding cytochrome c1), COX4I1 (gene encoding COX4I1 subunit of cytochrome c oxidase), ATP50 (gene encoding ATP50 subunit of ATP synthase), DLAT (gene encoding dihydrolipoamide-S-acetyltransferase, E2 subunit of PDH) and CS (gene encoding citrate synthase) was performed using an ABI PRISM 7500 instrument (Applied Biosystems, Foster City, CA, USA) with TaqMan® Universal PCR Master Mix, NO AmpErase® UNG and specific TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). PCR amplifications were performed at least in duplicate in a total reaction volume of 20 μL. The expression of beta-2-microglobulin (B2M) was used to compensate for variations in input RNA amounts and the efficiency of reverse transcription. The modified formula 2−∆Ct was used to calculate relative gene expression.
8
Psoralen-Induced Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 48 h exposure to different concentrations of psoralen (0.01, 0.05, 0.1, and 0.5 μmol/L), cells were collected for the extraction of total RNA by TRIzol (Invitrogen, Carlsbad, CA). The concentration and purity of RNA were determined using a ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE). Reverse transcription of RNA into cDNA was carried out using PrimeScript RT reagent kit with gDNA Eraser (Takara, Bio Inc., Shiga, Japan) according to the instructions of the manufacturer. Then, qPCR was performed with SYBR® Green PCR master mix (Takara, Bio Inc., Shiga, Japan) using standard protocols on an ABI PRISM 7500 instrument (Applied Biosystems, United States). The GAPDH was chosen as a reference. The amplification conditions were 5 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The 2−ΔΔCt method was applied to determine relative expressions of MMP-2 and MMP-9. Primers were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), and their sequences were presented in Supplementary Table S1 .
9
Quantification of NF-I and HIV-1 Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the cells using the RNAzol-RT kit (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturerss instructions. RNA concentration was measured using a Nanodrop spectrophotometer. First-strand cDNA was synthesized from 5 µg of total RNA using a SuperScript III first-strand synthesis kit (Invitrogen). Quantitation of NF-IA, NF-IB, NF-IC, and NF-IX, HIV-1 gag, and GAPDH were done using the following primers (NFI-A, Forward-CAGCCAAGTGACGCTGACA, Reverse-CCTCATTGCTCCTGGACTCAT; NF-1B, Forward-GCCACAATGATCCTGCCAAGAA, Reverse-GGTGGAGAAGACAGAGACCTCTGA; NFI-C, Forward-GGACAGGGATGGGCTCTG, Reverse-CGTTCTTCTGAGGCCAGTGC; NFI-X, Forward-CCACTGCCCAACGGACACTT, Reverse-CCGGGATAGAACACGTCATCA; HIV-1 gag Forward-CTAGGAACGATTCGCAGTTAATCCT, Reverse-CTCTATTGTGTGCATCAAAGGATAG; GAPDH Forward-GACAGTCAGCCGCATCTTCT, Reverse-TTAAAAGCAGCCCTGGTGAC).
Real-time PCR was performed on ABI Prism 7500 instrument (Applied Biosystems, Foster City, CA, USA) with a quantitect SYBR green kit (Qiagen, Valencia, CA, USA). The expression levels were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Real-time PCR was performed on ABI Prism 7500 instrument (Applied Biosystems, Foster City, CA, USA) with a quantitect SYBR green kit (Qiagen, Valencia, CA, USA). The expression levels were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
10
Quantifying Treg and Th17 Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA samples were divided into two parts, one batch for mRNA expression analysis of key factors in Treg and Th17 cell development and the second for the miRNA expression assay. A universal cDNA synthesis kit (Exiqon, Denmark) was used for cDNA synthesis of miR-223, with RNU48 as the reference gene (32 (link)) through a poly A tailing manner based on the manufacturer’s leaflet. Pre-designed specific primers of miR-223 and RNU48 for qRTPCR were supplied by Pars Genome Company (Tehran, Iran). An ABI PRISM 7500 instrument (Applied Biosystems, USA) was used for the qRT-PCR analysis. All reactions were performed in triplicate using standard protocols. CDNA synthesis of key factors TGF-β, INTERLEUKIN 23R (IL23R) and IL17a was performed with a RevertAid First Strand cDNA synthesis Kit (Thermo Scientific, USA) according to the manufacturer’s protocol. The expression level of each gene was normalized vs. 18srRNA in the same sample. All measurements were performed for three independent replicates.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!