Genomic DNA was isolated from peripheral blood leucocytes (Puregene, DNA purification system, Gentra Systems) and was stored at 4 °C until use. Identification of the allelic variants MTHFR 677C>T, MTHFR 1298A>C, MTR 2756A>G, TCN2 67A>G and TCN2 776C>G was performed by PCR-RFLP assay using HinfI, MboII, HaeIII, RsaI and ScrFI endonucleases, respectively [16 (link),30 (link),35 (link)]. After restriction enzyme digestion, PCR products were evaluated by gel electrophoresis analysis. Allelic discrimination of MTRR 66A>G variants was performed by real-time PCR (iQ5 Real-Time PCR Thermocycler, BioRad, California, USA) using TaqMan probes (TaqMan® SNP Genotyping Assays, Applied Biosystems, Waltham, MA, USA).
Iq5 real time pcr thermocycler
Manufactured by Bio-Rad
Sourced in United States
The IQ5 real-time PCR thermocycler is a laboratory instrument used for the amplification and detection of DNA or RNA sequences. It is capable of performing real-time quantitative PCR (qPCR) analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Complementary dna reverse transcription kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Qscript one step qrt pcr kit, by Quanta Biosciences (1 mentions)
Sybr green qpcr master mix reagent, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Nanophotometer p class, by Implen (1 mentions)
Chromatin ip dna purification kit, by Active Motif (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Chip it express enzymatic kit, by Active Motif (1 mentions)
6 protocols using iq5 real time pcr thermocycler
1
Genotyping of Folate Pathway Genes
2
Kidney RNA Extraction and Real-Time PCR
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Total RNA was extracted from the kidney tissue samples with TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed using a complementary DNA reverse transcription kit (Takara Bio, Tokyo, Japan). Reactions were performed in an iQ5 real-time PCR thermocycler (Bio-Rad, Hercules, CA, USA) using SYBR green as the fluorescent dye. The mRNA expression of the target genes was normalized to the control gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the comparative threshold cycle (2-ΔΔCT) method. S1 Table lists the primer sequences used in the real-time PCR experiments.
3
Quantifying PGC-1α Expression in Cardiac Myocytes
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Cardiac myocytes were processed with GeneJet RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) for extraction of total RNA. In total, 25 ng of extracted total RNA was used for all reactions and processed using a qScript One Step qRT PCR Kit (Quanta Biosciences, Beverly, MA, USA). Bio-Rad iQ5 real-time PCR thermocycler was used for amplification and analysis, with normalization to Gapdh, and relative gene expression calculated (2−ΔΔCT method). Primer sequences were PGC-1α-forward 5′-AAGTGTGGAACTCTCTGGAACTG-3′, PGC-1α-reverse 5′-GGGTTATCTTGGTTGGCTTTATG-3′, Gapdh-forward 5′-TGCACCACCAACTGCTTAGC-3′, and Gapdh-reverse 5′-GGCATGGACTGTGGTCATGAG-3′.
4
Quantitative RT-PCR for Angiogenic Factors
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Total RNA was extracted from tissue samples using Trizol reagent (Invitrogen), and the IQ5 real‐time PCR thermocycler (Bio‐Rad) was used to perform the qRT‐PCR. mRNA levels of VEGF‐A, FGF2 and PDGF‐BB were quantified with SYBR Green qPCR master mix reagent (Takara Biotechnology). The results were normalized against glyceraldehyde 3‐phosphate dehydrogenase expression and calculated using the 2−ΔΔCT method. Primers are listed in Table S2 .
5
Quantitative Real-Time PCR Analysis
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Cells were grown in 12-well plates, treated accordingly, and total mRNA isolated with RNeasy (QIAGEN). RNA concentration and purity were quantified with the NanoPhotometer P-Class (Implen). Then 2 μg of RNA was reverse transcribed in a 20-μL reaction using QuantiTect Reverse Transcription Kit (QIAGEN). Gene expression was assayed by TaqMan quantitative RT-PCR on the IQ5 real-time PCR thermocycler (Bio-Rad Laboratories). Gene expression was quantified using comparative threshold cycles (CT) in triplicate samples at 50 ng of RNA/well. Then 20-μL reactions were prepared using 2× universal TaqMan Master Mix and 1 μL of a gene-specific TaqMan assay (Applied Biosystems): rGH (Rn01495894_g1), human SSTR3 (Hs00265633_s1), rat SSTR2 (Rn01464950_g1), or rat Pit-1 (Rn00564562_m1). Amplicons were detected using probes tagged with MGB quencher and FAM dye. A rat β-actin control gene expression assay with MGB and VIC dye (4352340E; Applied Biosystems) was used as a reference control. Thermal cycling conditions were 95°C for 10 minutes; 95°C for 15 seconds, and 60°C for 1 minute for 40 cycles. Standard curves were generated for each probe ranging from 0.02 to 200 ng of cDNA.
6
Chromatin Immunoprecipitation Protocol
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Stable transfectants were seeded at 5 × 106/15-cm plate for 72 hours and chromatin isolated with a ChIP-IT Express Enzymatic kit (Active Motif). Chromatin concentration was established after phenol chloroform extraction, and 25 μg of total chromatin added to each ChIP reaction. Immunoprecipitated chromatin was purified for quantitative PCR with the Chromatin IP DNA Purification Kit (Active Motif). For quantification of chromatin recovery, each ChIP reaction was compared with a 10-fold dilution of input chromatin. Quantitative real-time PCR was performed with SYBR Premix Ex Taq II (Takara) on the IQ5 real-time PCR thermocycler (Bio-Rad Laboratories). Values are reported as percent recovery of total input chromatin. rGHP-F and rGHP-R primers were used to detect the first kilobase of the rGH promoter.
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