Total RNA was extracted from A549 or A549/DDP cells using TRIzol reagent (Sangon Biotech) according to the manufacturer's protocol. Then, the RNA was reverse transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR was performed using cDNA primers specific for mRNA. The gene GAPDH was used as an internal control. All the real-time PCR reactions were performed using Takara′s SYBR Premix Ex Taq™ II (Tli RNaseH Plus) in Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The 2-△△Ct method was used for quantification and fold change for target genes was normalized by internal control.
Biosystems 7500 fast real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Biosystems 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 thermal cycling to amplify and detect nucleic acid sequences in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Sybr premix ex taq 2 tli rnaseh plus, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Sangon (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Mircute mirna isolation kit, by Tiangen Biotech (1 mentions)
Cel mir 39, by Thermo Fisher Scientific (1 mentions)
Mircute plus mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Reaction buffer, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taqtm 2 tli rnaseh plus, by Takara Bio (1 mentions)
Prism 7, by GraphPad (1 mentions)
Rnasimple total rna extraction kit, by Tiangen Biotech (1 mentions)
7 protocols using biosystems 7500 fast real time pcr system
1
Quantitative RT-PCR Analysis of Gene Expression
2
Quantifying Toxoplasma gondii in Genomic Samples
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Genomic DNA was extracted from the collected samples using the DNeasy® Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. Parasite quantification was carried out by qPCR using primer pairs for the 529-bp repetitive element of T. gondii [37 (link)]. DNA samples were adjusted to 20 ng/μL, and reactions were performed in a final volume of 25 μL using GoTaq® qPCR Master Mix (Promega, Alcobendas, Madrid, Spain), 10 pmol of each primer and 100 ng of DNA in an Applied Biosystems 7500 FAST Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Amplification was performed by a standard protocol (10 min at 95 °C, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min). The number of T. gondii tachyzoites was calculated by interpolating the average Ct values on a standard curve equivalent to 1 × 105 − 1 × 10−1 tachyzoites generated by tenfold serial dilutions of parasite DNA in a solution of ovine genomic DNA at 20 ng/μL. Parasite proliferation was expressed as the parasite number/ng of DNA. Standard curves for T. gondii showed an average slope always close to −3.3 and an R2 > 0.98.
3
Quantitative Analysis of miRNA and mRNA
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Total RNA was extracted and reverse transcribed to cDNA using a miRcute miRNA Isolation Kit and a miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech, CAT. hz130911–251, China) following standard protocols. PCR was performed on a Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The relative expression level of miR-567 was normalized to that of U6 and the expression of ATG5 mRNA was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). miRNAs in exosomes were normalized using cel-miR-39 (Applied Biosystem, CA, USA) as an external control. The primer sequences were provided in Supplementary Table S2 .
4
qPCR Gene Expression Analysis Protocol
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Cells were seeded in 6-well plates in densities described in Section 2.2 . After 24 h, treatments were introduced—DMSO (vehicle), GW3965 (10 μM), and 1E5 (10 μM). Total RNA was extracted after 48 h of incubation using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA, #74104). cDNA was synthesized using 1 µg of total RNA with an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA, #1725035). qPCR reactions were set up to be 10 µL in total volume, including (0.5 µL forward primer, 0.5 µL reverse primer, and 1 µL nuclease-free water (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA, #AM9906) with 5 µL of SYBR Green (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA, #A25742), and 3 µL of 1:10 diluted cDNA. Reactions were loaded to MicroAmp™ Optical 96-Well GPLE Reaction Plates and quantified using a Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA, #4481192). Primers for the study were ordered from the PrimerQuest™ program, IDT, Coralville, Iowa, USA, and the list is provided in Supplementary Materials (see Figure S1) .
5
Genotyping of IGF1 and INS SNPs
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Genotyping of SNP rs35767 at the IGF1 gene locus was performed with TaqMan allelic discrimination using Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Courtaboeuf, France). SNP genotyping assay (ID: C_7999146_10) was purchased from Life technologies (Saint Aubin, France).
Genotyping of SNP rs689 at the INS gene locus was determined by the analysis of PCR products [30 (link)]. PCR amplification was in 96-well microliter plates, each 50 μl reaction contained DNA (100 ng), MgCl2 (1.5 mM), 10× reaction Buffer (Thermo Scientific), dNTPs (2.0 mM each), primers (1 μM each), and Taq Polymerase (1.25 U, Thermo Scientific, Saint Aubin, France). Amplified PCR products were digested with 1 unit of Hph1 enzyme (Thermo Scientific, Saint Aubin, France).
For genotyping quality, negative controls were included in each PCR plate. Twenty percent of samples were analyzed as duplicate for genotyping determination. The Hardy-Weinberg equilibrium (HWE) was calculated by computing the test for deviations in HWE and was shown to be present across genotypes. Allele frequencies were calculated and tested by test.
Genotyping of SNP rs689 at the INS gene locus was determined by the analysis of PCR products [30 (link)]. PCR amplification was in 96-well microliter plates, each 50 μl reaction contained DNA (100 ng), MgCl2 (1.5 mM), 10× reaction Buffer (Thermo Scientific), dNTPs (2.0 mM each), primers (1 μM each), and Taq Polymerase (1.25 U, Thermo Scientific, Saint Aubin, France). Amplified PCR products were digested with 1 unit of Hph1 enzyme (Thermo Scientific, Saint Aubin, France).
For genotyping quality, negative controls were included in each PCR plate. Twenty percent of samples were analyzed as duplicate for genotyping determination. The Hardy-Weinberg equilibrium (HWE) was calculated by computing the test for deviations in HWE and was shown to be present across genotypes. Allele frequencies were calculated and tested by test.
6
Rat Liver RNA Expression Analysis
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Total RNA was extracted from rat liver tissues with TRIZOL reagent (Tiangen, China). The concentration and purity of different RNA samples were evaluated using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States).The cDNA was synthesized by reverse transcriptase (Tiangen, China). Real-time qPCR was performed using the Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific (China) Co., Ltd.). The primers of real-time qPCR were used to amplify transcripts for c-Met, Irs1, Akt1, Pik3r1, Foxo1, Sirt1 and G6pc (Table 1 ). ß-actin served as the endogenous housekeeping gene to normalize the expression of the RNA sample levels. Relative gene expression levels were calculated using the 2−ΔΔCT method.
7
Quantitative Analysis of CD36 Expression
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Total cellular RNA was extracted using an RNA simple total RNA extraction kit (TIANGEN, China, Cat. No. DP419) according to the manufacturer’s instructions, and the concentration was measured. Reverse transcription of 500 ng total RNA was performed using Prime ScriptTM RT Master Mix (Perfect Real Time) (TaKaRa, Japan, Cat. No. RR036B). RT-qPCR was performed using TB Green Premix Ex TaqTM II (Tli RNaseH Plus) (TaKaRa, Japan, Cat. No. RRB20A) kit and applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, America), with the following primers: for CD36, 5'-CCAGTTGGAGACCTGCTTATC-3' and 5'-TCTGTAAACTTCTGTGCCTGTT-3'; and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ATGACATCAAGAAGGTGGTG-3' and 5'-CATACCAGGAAATGAGCTTG-3'. The relative fold-change was determined using the double delta method (2−ΔΔCT) normalized to GAPDH level and data analysis was performed using Prism7 software (GraphPad Software, Inc., California, USA).
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