Reverse transcription of 1 μg (with the exception of few cases with limited RNA amount) of RNA to cDNA was performed using the High-Capacity Kit (Applied Biosystems, Carlsbad, CA, USA). Gene expression analysis of HLA-C (Hs03044135_m1) and GAPDH (Hs03929097_g1), used as endogenous gene, was performed by TaqMan Gene Expression Assays (FAM-MGB) (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The assays were conducted in triplicate and the experiment was conducted on a DNA Engine Opticon 2 Real-Time Cycler (Bio-Rad, Hercules, CA, USA) using the following PCR conditions: initial activation 95°C for 10 minutes, 50 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute.
Dna engine opticon 2 real time cycler
Manufactured by Bio-Rad
Sourced in United States
The DNA Engine Opticon 2 Real-Time Cycler is a laboratory instrument designed for real-time PCR (polymerase chain reaction) analysis. It is capable of monitoring DNA amplification in real-time during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (3 mentions)
High capacity kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (1 mentions)
Genelute mammalian genomic dna miniprep kit, by Merck Group (1 mentions)
Mircute plus mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
First strand cdna synthesis kit, by Qiagen (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Ncode sybr green mirna qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using dna engine opticon 2 real time cycler
1
Gene Expression Analysis of HLA-C and GAPDH
2
Quantitative Real-Time PCR Analysis
3
Quantifying Gene Expression in B-CLL
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1μg RNA/sample was retro-transcribed using the High Capacity Kit (Applied Biosystems, Carlsbad, CA, USA). Gene expression analysis was performed by qRT-PCR, conducted on a DNA Engine Opticon 2 Real-Time Cycler (Bio-Rad, Hercules, CA, USA), using iQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) for each gene tested and for the reference genes. Actin Beta (ACTB) gene, which is one of the most often used reference genes in B-CLL gene expression studies [48 (link), 49 (link)], was used as reference gene. A subgroup of tumoral and non-tumoral samples was also analyzed using Glucuronidase Beta (GUSB) as reference gene.
Primers used in this study to conduct qRT-PCR are the following:
- 5’-AGACCATCAGTGCAAGCGAA-3’ (SHANK1 forward)
- 5’-GGGATCGAAGCTCGACTCAG-3’ (SHANK1 reverse)
- 5’-AAATCTGGCACCACACCTTC-3’ (ACTB forward)
- 5’-AGCACAGCCTGGATAGCAAC-3’ (ACTB reverse)
- 5’-CACCTAGAATCTGCTGGCTACT-3’ (GUSB forward)
- 5’-AGAGTTGCTCACAAAGGTCACA-3’ (GUSB reverse)
Gene expression data were analyzed using the ΔΔCT method. We used a t-test for independent series to compare the average ΔCT of CLL cases and controls.
Primers used in this study to conduct qRT-PCR are the following:
- 5’-AGACCATCAGTGCAAGCGAA-3’ (SHANK1 forward)
- 5’-GGGATCGAAGCTCGACTCAG-3’ (SHANK1 reverse)
- 5’-AAATCTGGCACCACACCTTC-3’ (ACTB forward)
- 5’-AGCACAGCCTGGATAGCAAC-3’ (ACTB reverse)
- 5’-CACCTAGAATCTGCTGGCTACT-3’ (GUSB forward)
- 5’-AGAGTTGCTCACAAAGGTCACA-3’ (GUSB reverse)
Gene expression data were analyzed using the ΔΔCT method. We used a t-test for independent series to compare the average ΔCT of CLL cases and controls.
4
Quantifying Transposon Copy Number by qPCR
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Genomic DNA was extracted from a pool (8-10) of whole flies using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, Saint Louis, Missouri, USA). qPCR Genomic copy number of telomeric (HET-A, TART and TAHRE) retrotransposon was estimated by qPCR conducted using DNA Engine Opticon 2 Real-Time Cycler (Bio-Rad, Hercules, CA, USA). qPCR reactions were conducted in a total volume of 20 µl containing: 20 ng genomic DNA, 10 µl iQ™ SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) and 6 pmol of each primer. Primer sequences were obtained from Walter et al. (21) . Reactions were performed using the following conditions: 3 min at 95°C followed by 40 cycles with 10 sec at 95°C and 1 min at 58°C. Experiments were conducted in triplicate. Threshold cycle values (Ct) were normalized against RpS17 copy number. The generated data were analyzed applying ΔΔCT method.
5
Quantifying miRNA Expression with qRT-PCR
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Each qRT-PCR included 0.8 μl miR-210/miR-16-1 primer set (5 μM ) (miR-210, forward: 5′-TTGACCTGTGCGTGTGACA-3′, reverse: 5′-TATGGTTGTTCTGCTCTCTGTCTC-3′ miR-16-1, forward: 5′-CGCCTGTAGCAGCACGTAA-3′, reverse: 5′-CAGAGCAGGGTCCGAGGTA-3′ Genepharma), 20 μl RT-PCR master mix (Genepharma), 0.2 μl Taq DNA polymerase (5 U μl−1; Genepharma), 4 μl of cDNA, and water to a final volume of 40 μl. PCR was performed on the DNA Engine Opticon 2 Real-Time Cycler (MJ Research, Inc., Waltham, MA, USA) with the following cycle profile: 95 °C for 3 min, followed by 45 cycles of 95 °C for 12 s, and 62 °C for 40 s. Cycle threshold (Ct) values were determined, and the expression levels were calculated in triplicate from the equation 2−ΔCt where the raw data of the target miRNA were normalised to the Ct of miR-16-1, which served as the internal control (Li et al, 2013b (link); Zanutto et al, 2014 (link)). PCR products were run on agarose gels to determine size, and dissociation curves were subsequently utilised to examine specificity of the qPCR assay.
6
miRNA cDNA Synthesis Protocol
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cDNA was synthesized using the miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech) on the DNA Engine Opticon 2 Real-Time Cycler (MJ Research, Inc.; Waltham, MA, U.S.A.). Each reaction mixture utilized 2× miRNA RT Reaction Buffer (10 μl), 2 μl miRNA RT Enzyme Mix, 3 μl total RNA, and 5 μl RNase-Free ddH2O to a reaction volume to 20 μl. Then, with the following cycle profile: 42°C for 60 min and 95°C for 3 min. Synthesized cDNA was stored at −20°C for further analysis.
7
NUTM2A-AS1 Expression Analysis in Cells
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RNAiso Plus Kit was applied to extract gross RNA from cells. The RNA was reverse transcribed into cDNA with First Strand cDNA Synthesis Kit (Qiagen, Germany). The cDNA templates were amplified by real-time PCR with Takara SYBR Green PCR Kit. GAPDH was utilizing as internal control. Real-time PCR was conducted via DNA Engine Opticon 2 Real-Time Cycler (MJ Research, Inc.). The relatively fold-change mRNA levels were computed. From Sangon Biotech, GAPDH and NUTM2A-AS1 primers were purchased. The following primer sequences were used for qPCR: GAPDH forward, 5′-TTTGGTATCGTGGAAGGACTC−3′ and reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′; NUTM2A-AS1 forward, 5′-TACCTCTAGTTCTTCCCGGC-3′ and reverse, 5′-TTTTGCTTTTCTCCTGGCCC-3′.
8
Synthesize miRNA cDNA from Total RNA
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cDNA specific for miRNA and the external control was synthesized from total RNA in a reaction mixture containing Escherichia coli Poly(A) Polymerase (5 units/μl) (Tiangen Biotech Co., Ltd.; Beijing, China), 5× rATP solution and 10× Poly(A) Polymerase buffer using the DNA Engine Opticon 2 Real-Time Cycler (MJ Research, Inc.; Waltham, MA, U.S.A.) with incubation at 37ºC for 60 min. Synthesized cDNA was stored at –80ºC for further analysis.
9
Quantitative analysis of miRNA expression
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cDNA specific for miR-210 and miR-16-1 (internal control) was synthesised from 3 to 4 μg of total RNA in a reaction mixture containing miR-210 (5′-GTCTGTATGGTTGTTCTGCTCTCTGTCTCATCCCTATCTACAACCATACAGACTCAGCCGCTG-3′)/miR-16-1 (5′-GTCGTATCCAGAGCAGGGTCCGAGGTACACGTTCGCTCTGGATACGACCGCCAATATT-3′) RT primers (Genepharma; Shanghai, China) and M-MLV reverse transcriptase on the DNA Engine Opticon 2 Real-Time Cycler (MJ Research, Inc.; Waltham, MA, USA) with the following cycle profile: 16 °C for 30 min, 42 °C for 30 min, 85 °C for 10 min. Synthesised cDNA was stored at −80 °C for further analysis.
10
FOXP1 Overexpression in VSMCs
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The coding sequence of FOXP1 was amplified using PCR and inserted into pcDNA3.0 (Invitrogen; Thermo Fisher Scientific, Inc.). PCR amplification was performed as follows: 94°C for 3 min, followed by 30 cycles of 94°C for 40 sec, 56°C for 45 sec and 72°C for 60 sec, followed by terminal elongation. A DNA Engine Opticon 2 Real-Time Cycler (MJ Research, Inc.) and Taq DNA Polymerase (Invitrogen; Thermo Fisher Scientific, Inc.) were used. Insertion accuracy was confirmed via direct Sanger sequencing. The construct was subsequently transfected into VSMCs using Lipofectamine 2000.
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