Total RNA was extracted using the Sepasol (Nacalai Tesque), and level of mature miRNAs was detected using TaqMan MicroRNA systems (Applied Biosystems) using primer specific for each mature miRNA supplied by Applied Biosystems using Light Cycler 1.5 (ROCHE). Briefly, a total of 500 ng RNA were reverse-transcribed with Taqman Reverse-Transcription PCR Kit with specific primer for miR-142-3p. Then, cDNA was mixed with TaqMan Universal Master Mix (Applied Biosystems) and was subjected for real-time PCR. Ct value was analyzed with SDS 2.4 and RQmanager 1.2.1 and quantitated using 2−ΔΔCt method (Livak, 2001). All data were normalized to endogenous control, the U6 snRNA. Sequences of the primers are T/brachyury 5′-cacaccactgacgcacacggt-3′, 5′-atgaggaggctttgggccgt-3′, Gata4 5′-agccggtgggtgatccgaag-3′, 5′-agaaatcgtgcgggagggcg-3′, Fgf5 5′-gcagtccgagcaaccggaact-3′, and 5′-ggacttctgcgaggctgcga-3′. For quantification of mRNA, total RNA (1 μg) from each sample was used to generate cDNA using ReverTra Ace qRT-PCR RT Kit (Toyobo). Then, cDNA was mixed with Sybr Green Master Mix (ROCHE) and was subjected for real-time PCR using Light Cycler 1.5 (ROCHE). Expression levels of mRNA were compared to known standard samples and normalized to GAPDH.
Lightcycler 1
Manufactured by Roche
Sourced in Germany, Switzerland, United States, Japan, United Kingdom, Finland, Italy
The LightCycler 1.5 is a real-time PCR instrument designed for quantitative nucleic acid analysis. It utilizes fluorescence detection to monitor the amplification of DNA or RNA samples during the PCR process. The LightCycler 1.5 provides accurate and reliable real-time PCR results.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (23 mentions)
Sybr premix ex taq, by Takara Bio (17 mentions)
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Trizol, by Thermo Fisher Scientific (12 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (11 mentions)
Quantitect sybr green pcr kit, by Qiagen (8 mentions)
Superscript 2 reverse transcriptase enzyme, by Thermo Fisher Scientific (7 mentions)
Quantitech sybr green pcr kit, by Qiagen (7 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Superscript 2, by Thermo Fisher Scientific (6 mentions)
Revertra ace qpcr rt master mix, by Toyobo (6 mentions)
Thunderbird sybr qpcr mix, by Toyobo (6 mentions)
Lightcycler faststart dna master sybr green 1, by Roche (6 mentions)
Quantitect reverse transcription kit, by Qiagen (6 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Faststart dna master sybr green 1, by Roche (6 mentions)
Lightcycler faststart dna masterplus sybr green 1 kit, by Roche (5 mentions)
Lightcycler rna master sybr green 1, by Roche (5 mentions)
Lightcycler faststart dna master sybr green 1 kit, by Roche (4 mentions)
Lightcycler 480 2, by Roche (4 mentions)
224 protocols using lightcycler 1
1
Quantitative Analysis of miRNAs and mRNA Levels
2
Quantitative Real-Time PCR Analysis of Mouse Aortic VSMC RNA
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Total RNA was extracted from the mouse aortic VSMCs using a RNA extraction kit (MachereyNagel) according to the manufacturers’ instructions. RNA was reverse transcribed to cDNA using the Moloney murine leukemia virus reverse transcriptase (M-MLV RT). Quantitative real-time PCR was performed in a LightCycler 1.2 (Roche) using the SYBR Green– based method for detection of gene amplification according to the manufacturer’s instructions. Amplification of the 60S ribosomal protein L32 (RPL32) or GAPDH served as an internal standard. The primer sequences used for amplification are listed in Table1 .
3
Evaluating Gene Expression in Cells
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Cells were seeded at 47,000 cells/9.5 cm2 on collagen‐I‐coated 6‐well plates (Corning, New York, USA) and incubated in different media (Table 1 ) for 3 days at 37℃. Total RNA from cultured cells was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription was carried out with the Reverse Transcription System (Promega). Alterations in gene expression were analysed by quantitative real‐time PCR (RT‐PCR) using the LightCycler FastStart DNA Master SYBR Green I kit (Roche) with the LightCycler 1.2 (Roche). Samples were run in duplicate using the following primers (Table S1 ): glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), aldehyde dehydrogenase family 3 member A1 (ALDH3A1), collagen 8A2 (Col8A2), lumican (LUM) and α‐smooth muscle actin (SMA). Relative fold changes in gene expression were analysed using the comparative CT (2 ‐ Δ Δ C T 35 Relative fold changes were calculated to the reference of CSK 0.5% FBS.
4
Quantitative gene expression analysis
5
Porcine mtDNA Quantification by qPCR
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The PCR mixture (20 μl) that was used to target porcine mitochondrial DNA sequences contained the following components: 1X Premix Ex Taq Master Mix (Perfect Real Time; TaKaRa, Dalian, China), 200 nM of forward primer (SW-171), 200 nM of reverse primer (SW-172), 50 nM of TaqMan probes (SW-301), 1 μl of each FFPE DNA solution, and 1 ng of porcine gDNA. The primer and probe sequences are listed in Table 1 . In order to normalize the quantification cycles (Cq) for each sample that had been spiked with porcine gDNA, reaction mixtures that contained no PCR amplification inhibitors and only 1 ng of porcine gDNA and 50 ng of human genomic DNA were used. Reactions were performed on a LightCycler 1.2 (Roche, Indianapolis, IN, USA) using the following cycling conditions: an initial denaturation step at 95°C for 20 sec followed by 40 cycles of 95°C for 5 sec, and 60°C for 30 sec.
6
RT-PCR Detection of H7N9 Influenza Virus
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The H7N9 RT-PCR detection kit used in our study was from Shanghai Zhijiang Biotechnology Co. Ltd. and Guangzhou Da'an Biotechnology Co. Ltd. RNA was first extracted from specimens according to the manufacturer's instruction. A 25 μL reaction system was set up containing 2 μL template RNA, 1 μL 25× RT-PCR enzyme mix, 0.5 μL Probe (20 μM), 1 μL of each of the primers (10 μM), 12.5 μL of 2x RT-PCR master mixes, and 7 μL RNase free water. The test was performed using a LightCycler 1.2 (Roche, Germany). Amplification conditions were set as follows: 45°C for 5 min, 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 20 s.
7
Quantitative HIV-1 Viral Load and DNA Detection
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The HIV-1 viral load in the plasma of patients infected with HIV-1 was quantitatively detected using a standardized RT-qPCR (Cobas Amplicor HIV-1 Monitor Test; version 1.5; Ultra-sensitive specimen preparation; Roche Diagnostic Systems Inc) as previously described (17 (link)). The detection limit in the plasma was defined as 50 HIV-1 RNA copies/ml.
The total HIV-1 DNA, including the integrated HIV-1 DNA and episomal two long terminal repeat (2LTR) circles, in the peripheral blood was detected using a HIV-1 DNA Detection kit (PCR-Fluorescent Probing; SUPBIO; Guangzhou Hailite Biotechnology Co., Ltd.). Briefly, the total DNA was isolated from the blood using a QIAamp DNA Blood Mini Kit (Qiagen GmbH). The 50 µl reaction system contained 5 µl DNA and 45 µl PCR master mix. The test was performed using a Light Cycler 1.2 (Roche Diagnostics GmbH). The amplification conditions were set as follows: i) five cycles of 37°C for 5 min, 95°C for 10 min; ii) 95°C for 15 sec, 65°C for 15 sec and 72°C for 20 sec; iii) 40 cycles of 95°C for 15 sec, 62°C for 15 sec and 72°C for 20 sec; and iv) 10 cycles of 95°C for 15 sec, 52°C for 15 sec and 72°C for 32 sec. Two standard curves were calculated according to the volume of blood or PBMC counts. The results were expressed as copies/ml and copies/106 PBMCs.
The total HIV-1 DNA, including the integrated HIV-1 DNA and episomal two long terminal repeat (2LTR) circles, in the peripheral blood was detected using a HIV-1 DNA Detection kit (PCR-Fluorescent Probing; SUPBIO; Guangzhou Hailite Biotechnology Co., Ltd.). Briefly, the total DNA was isolated from the blood using a QIAamp DNA Blood Mini Kit (Qiagen GmbH). The 50 µl reaction system contained 5 µl DNA and 45 µl PCR master mix. The test was performed using a Light Cycler 1.2 (Roche Diagnostics GmbH). The amplification conditions were set as follows: i) five cycles of 37°C for 5 min, 95°C for 10 min; ii) 95°C for 15 sec, 65°C for 15 sec and 72°C for 20 sec; iii) 40 cycles of 95°C for 15 sec, 62°C for 15 sec and 72°C for 20 sec; and iv) 10 cycles of 95°C for 15 sec, 52°C for 15 sec and 72°C for 32 sec. Two standard curves were calculated according to the volume of blood or PBMC counts. The results were expressed as copies/ml and copies/106 PBMCs.
8
Temporal Gene Expression Profiling of Embryonic Stem Cell Differentiation
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Total RNA extraction from ES cells before differentiation, and EBs on day 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5 after differentiation, was performed using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). First-strand cDNA synthesis was performed using SuperScript II reverse transcriptase (Gibco-BRL). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed with a LightCycler SYBR Green I Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. Gene expression levels were determined by a LightCycler 1.2 (Roche, Basel, Switzerland). The qRT-PCR data were analyzed using the second derivative maximum method available with LightCycler Software Version 3.5.3. The sequences of the qRT-PCR primers for Rex1, Brachyury (Bra), Flk1, Nkx2.5, Cited4, and β-actin genes are listed in Table 1 . The β-actin gene was used as a reference molecule.
9
Gene Expression Analysis of Cultured Cells
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The cells were seeded at 9000 cells/1.8 cm2 on collagen-I-coated 24-well plates (Corning, New York, NY, USA) and incubated in different media (Table 2 ) for 48 h at 37 °C. The total RNA from cultured cells was extracted using RNeasy MiniKit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Reverse transcription was carried out with the Reverse Transcription System (Promega, Madison, WI, USA). Alterations in gene expression were analyzed by quantitative real-time PCR (RT-PCR) using the LightCycler FastStart DNA Master SYBR Green I kit (Roche) with the LightCycler 1.2 (Roche). The samples were taken in duplicate using the following primers (Supplementary Table S1 ): glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-smooth muscle actin (SMA), lumican (LUM) and aldehyde dehydrogenase family 3 member A1 (ALDH3A1). Relative fold changes in gene expression were analyzed using the comparative CT (2−ΔΔCT) method for 6 different donors [76 (link)]. Relative fold changes were calculated in comparison to the control group.
10
Quantification of Gene Expression in Fungal-Infected Plants
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RNA was extracted from detached leaves of barley cultivar Plaisant or rice cultivar Sariceltik inoculated with droplets of conidia of either isolate P1.2 or Δbip1 mutant 24 hai and 17 hai, respectively. Three independent replicate samples were harvested for each treatment and RNAs were extracted separately. Genomic DNA was removed using DNA-free (Ambion). Five μg of total RNA were reverse transcribed using the ThermoScript RT-PCR system (Invitrogen) according to the manufacturer’s instructions. The resulting cDNAs were diluted 10 times for analysis. Real-time PCR was carried out with the LightCycler Faststart DNA Master SYBR Green I kit (Roche Diagnostics) using a Light Cycler 1.2 (Roche Diagnostics). Primers used for qPCR are listed in S5 Table
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