Light cycler instrument

Manufactured by Bio-Rad

Sourced in United States

The Light Cycler instrument is a real-time PCR (Polymerase Chain Reaction) system designed for rapid and precise nucleic acid amplification analysis. It provides accurate temperature control and detection capabilities for quantitative and qualitative PCR applications.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (3 mentions) Primescript rt reagent kit, by Takara Bio (3 mentions) Microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions) Miscript 2 rt kit, by Thermo Fisher Scientific (2 mentions) Exiqon sybr green master mix, by Qiagen (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Sybr green pcr kit, by Enzynomics (1 mentions) Sybr premix ex taq 2 kit, by Takara Bio (1 mentions) Trizol reagent, by Merck Group (1 mentions) Taqman microrna reverse transcription kit, by Merck Group (1 mentions) Reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Icycler iq5 real time pcr detection system, by Bio-Rad (1 mentions) Ssofast evagreen supermix, by Bio-Rad (1 mentions) Iscript select cdna synthesis kit, by Bio-Rad (1 mentions) Mirneasy mini kit, by Qiagen (1 mentions) Iscript cdna kit, by Bio-Rad (1 mentions) Itaq sybr green master mix, by Bio-Rad (1 mentions) Quick rna kit, by Zymo Research (1 mentions) Sybr green, by Solarbio (1 mentions) Sybr primescript rt pcr kit, by Takara Bio (1 mentions)

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Bio-Rad cyclers (3 citations)

10 protocols using light cycler instrument

Quantitative RT-PCR Analysis of Gene Expression

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TRIzol reagent (Sigma-Aldrich, USA) was used to extract the total RNA from tissues and cells. Next, reverse transcription of RNA samples was performed to synthesize cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Sigma-Aldrich, USA), according to the manufacturer’s instructions. For PCR amplification, a Light Cycler instrument (Bio-Rad, Hercules, USA) was used to prepare the reaction system, followed by detection using the SYBR Green PCR kit (Enzynomics, Korea) and SYBR Premix Ex Taq II kit (Takara, USA). Relative expression was calculated using the 2^−ΔΔCt method, and normalized to glyceraldehyde‐3-phosphate dehydrogenase (GAPDH) and uracil 6 [19 (link)]. Primer sequences are listed in Table 1.

Table 1.

The sequences of the primers in this study

Primer	Sequences
miR-361-3p	Forward: 5’-UCCCCCAGGUGUGAUUCUGAUUU-3’
	Reverse: 5’-GCAAATCAGAATCACACCTG-3’
miR-3940-3p	Forward: 5’-CTCAAGGACCACCGCATC-3’
	Reverse: 5’-ATCTGCAAGGGACAGCACAG-3’
BBOX1-AS1	Forward: 5’-TGTGTGTTTCCTGAGGCCTC-3’
	Reverse: 5’-CGCCTCTCTTGGAACACCTT-3’
LAMC2	Forward: 5’-GCCTTTTGGCACCTGTATTC-3’
	Reverse: 5’-CAGGATTCTCATCCCCTGAA-3’
Bax	Forward: 5’-GGTTGCCCTCTTCTACTTT-3’
	Reverse: 5’-AGCCACCCTGGTCTTG-3’
Bcl-2	Forward: 5’-ACTTTGCAGAGATGTCCAGT-3’
	Reverse: 5’-CGGTTCAGGTACTCAGCAT-3’
GAPDH	Forward: 5’-CCATGTTCGTCATGGGTGTG-3’
	Reverse: 5’-GGTGCTAAGCAGTTGGTGGTG-3’
U6	Forward: 5’-CGCTTCGGCAGCACATATACTA-3’
	Reverse: 5’-TATGGAACGCTTCACGAATTTGC-3’

Zhao C., Shi W, & Chen M. (2022). Long non-coding RNA BBOX1-antisense RNA 1 enhances cell proliferation and migration and suppresses apoptosis in oral squamous cell carcinoma via the miR-3940-3p/laminin subunit gamma 2 axis. Bioengineered, 13(4), 11138-11153.

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Quantification of miR-27a and FBXW7 mRNA

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Total RNA was extracted from cells with the TRIzol reagent (Invitrogen, U.S.A.). Reverse transcription of miR-27a and F-box and WD-40 domain protein 7 (FBXW7) was synthesized using the miScript II RT kit and the reverse transcription kit (Invitrogen, Carlsbad, CA), respectively. miR-27a and FBXW7 expressions were measured using the Exiqon SYBR Green Master Mix (Exiqon, Vedbaek, Denmark) on a Light Cycler instrument (Bio-Rad). The primers used were as follows: miR-27a F: 5′-CCCAAGCTTACTGTGAAACTGTGAAACGTGAAACTGTGAAACTGTGAAACTGTGAATCTAGAGC-3′; miR-27a R: 5′-GCTCTAGATTTCA-3′; U6 F: 5′-TGCGGGTGCTCGCTTCGCAGC-3′; U6 R: 5′-CCAGTGCAGGGTCCGAGGT-3′; FBXW7 F: 5′- GTCCCGAGAAGCGGTTTGATA-3′, FBXW7 R: 5′-TGCTCAGGCACGTCAGAAAAG-3′; GAPDH F: 5′-AGGTCGGTGTGAACGGATTTG-3′, GAPDH R: 5′-TGTAGACCATGTAGTTGAGGTCA-3′. Relative quantification was determined by normalization to U6 or GAPDH. The qRT-PCR assays were performed in triplicate and the relative expression levels were calculated based on the 2^−ΔΔC_t method [13 (link)].

Guo Y., Zhang Z., Wang Z., Liu G., Liu Y, & Wang H. (2020). Astragalus polysaccharides inhibit ovarian cancer cell growth via microRNA-27a/FBXW7 signaling pathway. Bioscience Reports, 40(3), BSR20193396.

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Quantification of Gene Expression in Fungi

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Total RNA was purified as in previously reported protocols [69 (link)] and cDNA was synthesized using iScript Select cDNA Synthesis Kit (BioRad, Hercules, CA, USA). Primers for quantification of gene expression (Table S3) were designed using the quantPrime tool [70 (link)] and analyzed with the Primerselect program. For the housekeeping gene specific primers (Ubiquitine ligase, Ubiupper and Ubilower) were used [71 (link)]. The RT-qPCR was performed on the LightCycler Instrument (iCycler iQ5 real-time PCR detection system; Bio-Rad) using SsoFast EvaGreen Supermix (Bio-Rad). Mean comparisons were made by the Student’s t-test taking into consideration ammonium as the reference medium and wt as the reference strain. Then, statistical significance (p < 0.05) is highlighted between nitrate, nitrite or without N and ammonium medium of the same time and strain sample with a; and between nit2 mutant and wt of the same time and medium sample with b.

Gomez-Osuna A., Calatrava V., Galvan A., Fernandez E, & Llamas A. (2020). Identification of the MAPK Cascade and its Relationship with Nitrogen Metabolism in the Green Alga Chlamydomonas reinhardtii. International Journal of Molecular Sciences, 21(10), 3417.

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Quantification of miR-93 Expression in Lung Tissues

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Total RNA was isolated from the lung tissues or BALF using the miRNeasy mini kit (Qiagen, Inc.). The reverse transcription of miR-93 was performed using the miScript II RT kit and the reverse transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.), respectively. miR-93 expression was measured using Exiqon SYBR-Green Master Mix (Exiqon) on a Light Cycler instrument (Bio-Rad Laboratories, Inc.). The reaction mixtures were denatured at 95°C for 3 min, followed by 40 two-step cycles of 95°C for 10 sec and 60°C for 30 sec. The primers for RT-qPCR analysis were as follows: miR-93 forward, 5′-AGG CCC AAA GTG CTG TTC GT-3′ and reverse, 5′-GTG CAG GGT CCG AGG T-3′; U6 forward, 5′-TGC GGG TGC TCG CTT CGC AGC-3′ and reverse, 5′-CCA GTG CAG GGT CC GAG GT-3′. miRNA relative expression levels were analyzed using the 2^−ΔΔcq method (24 (link)) and determined by normalization to U6.

Gao H., Xiao D., Gao L, & Li X. (2020). MicroRNA-93 contributes to the suppression of lung inflammatory responses in LPS-induced acute lung injury in mice via the TLR4/MyD88/NF-κB signaling pathway. International Journal of Molecular Medicine, 46(2), 561-570.

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Quantifying HIV LTR Expression

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RNA was isolated using a Quick-RNA kit (Zymo), treated with DNase I, and cDNA synthesized with an iScript cDNA kit (BioRad). Semi-quantitative RT-PCR was performed to detect HIV LTR as previously described [29 (link)] using iTaq SYBR Green master mix (BioRad) on a LightCycler instrument (BioRad) and normalized to the GAPDH housekeeping gene.

Fan X., Xu J., Files M., Cirillo J.D., Endsley J.J., Zhou J, & Endsley M.A. (2019). Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing). Tuberculosis (Edinburgh, Scotland), 116, S28-S33.

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Quantifying miR-129 and TAK1 Expression

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Total RNA was isolated from the lung tissues using the RNeasy Mini kit (Qiagen GmbH). Reverse transcripts of miR-129 and TAK1 were synthesized using the MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) and the PrimeScript RT reagent kit (Takara Bio, Inc.), respectively. miR-129 and TAK1 expression was measured with SYBR-Green (Beijing Solarbio Science & Technology Co., Ltd.) on a Light Cycler instrument (Bio-Rad Laboratories, Inc.). The following primers were used for RT-qPCR analysis: miR-129 forward, 5′-GTTGGGGAGATTTAGTTTGTT-3′ and reverse, 5′-CCTACTCCAATTCCCCCTATAATAC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; TAK1 forward, 5′-GATATCCTGTCGACAGCCTCCGC-3′ and reverse, 5′-AACGTAACGGGCCCAGAGAA-3′; GAPDH forward, 5′-GTGGTGAAGACGCCAGTGGA-3′ and reverse, 5′-CGAGCCACATCGCTCAGACA-3′. The thermocycling conditions were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 10 min. Relative miRNA expression was analyzed using the 2^−ΔΔCq method (28 (link)).

Yao W., Xu L., Jia X., Li S, & Wei L. (2021). MicroRNA-129 plays a protective role in sepsis-induced acute lung injury through the suppression of pulmonary inflammation via the modulation of the TAK1/NF-κB pathway. International Journal of Molecular Medicine, 48(1), 139.

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Quantifying miR-144a-3p and EZH2 Expression in OSCC

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Total RNA was extracted from the OSCC tissues and cells using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Bio, Inc.) and the MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.), respectively. miR-144a-3p and EZH2 expression levels were measured using a SYBR^® PrimeScript™ RT-PCR kit (Takara Bio, Inc.) on a Light Cycler instrument (Bio-Rad Laboratories, Inc.). The primers of miR-144a-3p and EZH2 were as follows: miR-144-3p forward, 5′-GCC CCT ACA GTA TAG ATG ATG TA-3′ and reverse, 5′-GTG CAG GGT CCG AGG T-3′; U6 forward, 5′-GCT TCG GCA GCA CAT ATA CTA AAA T-3′ and reverse, 5′-CGC TTC ACG AAT TTG CGT GTC AT-3′; EZH2 forward, 5′-TTG TTG GCG GAA GCG TGT AAA ATC-3′ and reverse, 5′-TCC CTA GTC CCG CGC AAT GAG C-3′; GAPDH forward, 5′-GTG GTG AAG ACG CCA GT G GA-3′ and reverse, 5′-CGA GCC ACA TCG CTC AGA CA-3′. U6 and GAPDH were used as internal controls for detecting miR-144-3p and EZH2, respectively and fold changes were calculated using the 2^-∆∆Cq method (14 (link)).

He L., Liao L, & Du L. (2020). miR-144-3p inhibits tumor cell growth and invasion in oral squamous cell carcinoma through the downregulation of the oncogenic gene, EZH2. International Journal of Molecular Medicine, 46(2), 828-838.

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Quantification of Hepatic Gene Expression

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Total RNA was extracted from frozen liver using Trizol agent according to manufacturer’s instructions. The concentrations of RNA were measured with Nanodrop 2000 (Thermo Fisher, Waltham, MA, USA). Then 2 μg of total RNA was subjected to transcribe the cDNA. Real-time polymerase chain reaction (PCR) was performed with a light cycler instrument (BIO-RAD, Hercules, CA, USA) to analyze the mRNA expression levels of Nrf2, HO-1, glycogen synthase (GS), glycogen synthase kinase 3β (GSK-3β), glucokinase (GK) and SYBR green was used to detect the amplified products. The PCR cycle was as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s and extension at 72 °C for 15 s. The primers sequences for target genes were listed in Table 1. β-actin was amplified to normalize the quantification results of target gene expression using the 2^−ΔCt method.

Xu J., Zhang W., Lu Z., Zhang F, & Ding W. (2017). Airborne PM2.5-Induced Hepatic Insulin Resistance by Nrf2/JNK-Mediated Signaling Pathway. International Journal of Environmental Research and Public Health, 14(7), 787.

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Quantifying let-7e and SOCS1 Expression

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Total RNA was isolated from lung tissues or cells using RNeasy Mini Kit (Qiagen Corporation, Hilden, Germany). Reverse transcription of let-7e and SOCS1 (1 µg) was synthesized using the MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc) and the PrimeScript RT Reagent Kit (Takara Bio, Inc., Tokyo, Japan), respectively. Let-7e and SOCS1 expression were measured using the SYBR Green mixture (Solarbio, Beijing, China) on a Light Cycler instrument (Bio-Rad). The primers for qRT-PCR analysis were as follows: let-7e forward, 5′-AGCAAGCTTTGGCACCCACCCGTAGAAC-3′ and reverse, 5′-TAAGGATCCGATGCAGGGACAAGGACAGAA-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. SOCS1 forward, 5′-CTTCTGTAGGATGGTAGCACAC-3′, 5′-AGGAAGAGGAGGAAGGTTCT-3′; GAPDH R: 5´-GTGGTGAAGACGCCAGTGGA-3´; F: 5´-CGAGCCACATCGCTCAGACA-3´. The procedures of amplification reaction were performed as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 s and 60°C for 45 s, 30 min at 72°C. The miRNA relative expressions were analyzed using the 2^−ΔΔCT method [18 (link)].

Li W., Zhang W., Liu J., Han Y., Jiang H., Ji G, & Liu W. (2021). Down-regulation of miR-let-7e attenuates LPS-induced acute lung injury in mice via inhibiting pulmonary inflammation by targeting SCOS1/NF-κB pathway. Bioscience Reports, 41(1), BSR20201089.

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Quantifying miR-199a-3p and YAP1 Expression in Ovarian Cancer

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Total RNA was extracted from tissues and SKOV-3 and OV90 cells (1×10⁶) using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using the miScript II RT and RevertAid First Strand cDNA Synthesis kits (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. miR-199a-3p and YAP1 expression was measured using the Exiqon SYBR Green Master Mix (Exiqon; Qiagen GmbH) on a Light Cycler instrument (Bio-Rad Laboratories, Inc.). The reaction mixtures were denatured at 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The primers for RT-qPCR analysis were as follows: miR-199a-3p forward, 5′-TTTCTCGAGGAAGATGCTCACCAGCCCTTTA-3′ and reverse, 5′-TTTTCTAGAGCATCATCTTGCCAGCGACT-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; YAP1 forward, 5′-CGGTCCACTTCAGTCTCC-3′ and reverse, 5′-GAGTGTGGTGGACAGGTACTG-3′; GAPDH forward, 5′-GTGGTGAAGACGCCAGTGGA-3′ and reverse, 5′-CGAGCCACATCGCTCAGACA-3′. The expression levels of miR-199a-3p and YAP1 were normalized to those of of U6 and GAPDH, respectively. RT-qPCR assays were performed in triplicate and the relative expression of each gene was calculated using the 2^−∆∆Cq method (15 (link)).

He Y., Yu X., Tang Y., Guo Y., Yuan J., Bai J., Yao T, & Wu X. (2021). MicroRNA-199a-3p inhibits ovarian cancer cell viability by targeting the oncogene YAP1. Molecular Medicine Reports, 23(4), 237.

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