Lightcycler system

Manufactured by Roche

Sourced in Germany, Switzerland, United States, Japan, France, United Kingdom, China

The LightCycler system is a real-time PCR platform designed for quantitative gene expression analysis and genotyping. It enables rapid thermal cycling and sensitive fluorescence detection, providing researchers with a tool for accurate and efficient nucleic acid amplification and quantification.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (68 mentions) Rneasy mini kit, by Qiagen (35 mentions) Trizol, by Thermo Fisher Scientific (34 mentions) Primescript rt reagent kit, by Takara Bio (27 mentions) Sybr premix ex taq 2, by Takara Bio (22 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (15 mentions) M mlv reverse transcriptase, by Promega (11 mentions) Rneasy kit, by Qiagen (10 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (10 mentions) Trizol reagent, by Takara Bio (10 mentions) Faststart dna master sybr green 1, by Roche (9 mentions) Thunderbird sybr qpcr mix, by Toyobo (9 mentions) Primescript rt reagent, by Takara Bio (8 mentions) Rnaiso plus, by Takara Bio (7 mentions) Superscript 3, by Thermo Fisher Scientific (7 mentions) Primescript rt kit, by Takara Bio (7 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (7 mentions) Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (7 mentions) Sybr green master mix, by Roche (7 mentions) Lightcycler 480 sybr green 1 master mix, by Roche (6 mentions)

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466 protocols using lightcycler system

qRT-PCR Assay for mRNA and miRNA Quantification

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For qRT-PCR of mRNA targets, total RNA was extracted from cancer cells using RNAiso Plus (TaKaRa, 09108B). cDNA synthesis was performed according to the manufacturer’s instructions (TaKaRa, DRR047A), and qRT-PCR was performed with SYBR Premix Ex Taq II (TaKaRa, DRR081A) using a LightCycler system (Roche). The PCR reaction conditions for all of the assays were 94 °C for 30 seconds, followed by 40 cycles of amplification (94 °C for 5 seconds, 60 °C for 30 seconds and 72 °C for 30 seconds). GAPDH mRNA was used to normalize RNA inputs. The qRT-PCR primers are listed in Additional file 1: Table S2.
For qRT-PCR of miRNAs, small RNAs were extracted from cancer cells or tumor tissues using RNAiso for small RNAs (TaKaRa, D340A). miRNAs were converted to cDNA using a cDNA synthesis kit (TaKaRa, DRR047A), and qRT-PCR was performed with SYBR Premix Ex Taq II (TaKaRa, DRR081A) using a LightCycler system (Roche). The PCR reaction conditions for all of the assays were 95 °C for 20 seconds, followed by 40 cycles of amplification (95 °C for 10 seconds, 60 °C for 20 seconds and 70 °C for 5 seconds). U6 was used to normalize the RNA inputs. All of the primers were from the Bulge-Loop™ miRNA qRT-PCR primer set (RiboBio, MQP-0102, China).

Zhang X., Jiang P., Shuai L., Chen K., Li Z., Zhang Y., Jiang Y, & Li X. (2016). miR-589-5p inhibits MAP3K8 and suppresses CD90+ cancer stem cells in hepatocellular carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 35, 176.

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Quantitative Real-Time PCR Protocol

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For mouse tissues, total RNA was prepared using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA was treated with DNase, and 1 μg of RNA was used for reverse transcription (RT). 20X diluted cDNA was used for RT-quantitative PCR (RT-qPCR) reactions. The RT-qPCR reactions were performed using the Light-Cycler system (Roche) and a qPCR Supermix (Qiagen) with the indicated primers. Total RNA from plant samples was prepared using TRIzol as previously described (Connolly et al., 2006 ). 2 μg of RNA was used for RT using QuantiTect reverse transcription kit (Qiagen). 20X diluted cDNA was used for RT-qPCR reactions using the Light-Cycler system (Roche) and a LightCycler 480 SYBR Green I Master mix (Roche). Ubiquitin gene UBQ10 (AT4G05320) was used as a reference. The average of at least three technical repeats was used for each biological data point. Primer sequences are shown in Table S1 and S2.

Moullan N., Mouchiroud L., Wang X., Ryu D., Williams E.G., Mottis A., Jovaisaite V., Frochaux M.V., Quiros P.M., Deplancke B., Houtkooper R.H, & Auwerx J. (2015). Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research. Cell reports, 10(10), 1681-1691.

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Viral RNA Extraction and SARS-CoV-2 Detection

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Swabs were briefly vortexed in 500 µL PBS and viral RNA was extracted from 140 µL in accordance with the manufacturer’s instructions (QIamp viral RNA; Qiagen, Toronto, ON, Canada). RNA extractions were performed within the 48 h following sampling (swabs were kept at 4 °C in the meantime). RT-qPCR was performed on 96-well plates with a final volume of 20 µL in a Light Cycler system (Roche, Penzberg, Germany). The mixes were prepared in accordance with the manufacturer’s instructions (QuantiNova Probe; Qiagen, Toronto, ON, Canada) with 2 µL of RNA and primers that targeted the E-gene. To obtain human-derived viral RNA and perform phylogenetic analysis, the owner from case 3 self-performed a nasal swab which was treated as a feline one. To check whether the material in the oropharyngeal and rectal swabs originated from felines, RT-qPCR targeting the feline 40S ribosomal protein S7 (RPS7) gene was also performed. SARS-CoV-2-positive swabs were further tested for human ribosomal protein L30 (RPL30) mRNA expression, to rule out human contamination. RPS7 and RPL30 RT-qPCR were performed on 96-well plates in a final volume of 10 µL in a Light Cycler system (Roche, Penzberg, Germany). Mixes were prepared according to the manufacturer’s instructions (QuantiFast; Qiagen, Toronto, ON, Canada). The primers’ sequences are listed in Supplementary Table S2.

Bessière P., Fusade-Boyer M., Walch M., Lèbre L., Brun J., Croville G., Boullier S., Cadiergues M.C, & Guérin J.L. (2021). Household Cases Suggest That Cats Belonging to Owners with COVID-19 Have a Limited Role in Virus Transmission. Viruses, 13(4), 673.

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Quantitative Analysis of Gene Expression

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Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's protocol, and subsequent reverse transcription was performed using a ReverTra Ace -α- reverse transcription kit (Invitrogen). Real-time PCR was performed according to the SYBR Premix Ex Taq kit (Takara, Shiga, Japan) protocol in a Roche LightCycler system (Roche, Basel, Switzerland). The primers utilized for the real-time PCR are as follows: p53: Forward 5′-TCA GCATCTTATCCGAGTGGAA-3′ Reverse 5′-TGTAGT GGATGGTGGTACAGTCA-3′ USP9X: Forward 5′-CAAT GGATAGATCGCTTTATA-3′ Reverse 5′-CTTCTTG CCATGGCCTTAAAT-3′ Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): Forward 5′-CGGAGTCAA CGGATTTGGTCGTAT-3′ Reverse 5′-AGCCTTCTCCA TGGTGGTGAAGAC-3′

Wang X., Bao Y., Dong Z., Chen Q., Guo H., Ziang C, & Shao J. (2017). WP1130 attenuates cisplatin resistance by decreasing P53 expression in non–small cell lung carcinomas. Oncotarget, 8(30), 49033-49043.

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RNA Extraction and qRT-PCR Analysis

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We used TRIzol reagent (Life Technologies, Carlsbad, CA, USA) with a standard procedure to extract and purify the total RNA from the tumor and adjacent normal tissues and cell lines. We used a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA) to confirm the quantity and quality of the extracted RNA. Then we used a reverse transcription kit (TaKaRa, Dalian, P.R. China) to synthesize complementary DNA (cDNA) according to the user guide of the kit. Briefly, we prepared a master mixture on ice, and the mixture contained 10 μl of SYBR Green qRT-PCR Master Mix (Qiagen, Hilden, Germany), 2 μl of cDNA sample, and 2 μl of primers. We added RNase-free water to the mixture to make it up to 20 μl. We performed all reactions in a Roche LightCycler system (Roche, Basel, Switzerland).

Wang K., Zhang M., Wang C, & Ning X. (2019). Long Noncoding RNA LINC01296 Harbors miR-21a to Regulate Colon Carcinoma Proliferation and Invasion. Oncology Research, 27(5), 541-549.

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RNA Isolation and Quantification by qPCR

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Trizol (Life Technologies, United States) was employed to isolate total cellular and tissue RNA in accordance with specific protocols. The content and purity of RNA were detected through a NanoDrop 2000 (Thermo Fisher Scientific, United States). To carry out mRNA quantification, we applied a reverse transcription kit (Vazyme, China) for the reverse transcription of total RNA according to specific instructions. Afterward, SYBR Green Master Mix (Vazyme, China) was utilized to perform qPCR, whereas a Roche Light Cycler system (Roche, Switzerland) was adopted for analysis, with GAPDH being an endogenous control. Table 2 lists the primer sequences. Gene expression was normalized to the GAPDH level, which was presented in the manner of FC (2−ΔΔCT). All results were repeated thrice.

Wang X.J., Qi Y.D., Guan H.C., Lin H.G., He P.Q., Guan K.W., Fu L., Ye M.Q., Xiao J, & Wu T. (2021). Gegen Qinlian Decoction Ameliorates Hyperuricemia-Induced Renal Tubular Injury via Blocking the Inflammatory Signaling Pathway. Frontiers in Pharmacology, 12, 665398.

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Quantitative RNA Expression Analysis in Plants

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These analyses were performed as previously described by Leba et al.^{17 (link)}. RNA extraction from leaves and reverse transcription were conducted as recommended by the manufacturers’ protocols (respectively with the Nucleospin RNA plant kit from Macherey-Nagel and the superscript reverse transcriptase II from Invitrogen). Quantitative PCR was run on a Roche lightcycler system (Roche Diagnostics) using specific pairs of primers (listed in Table S1). Each value obtained is an average of three independent biological replicates, and the experiment was repeated two times for each biological repeat. The measurements obtained for the reference gene, actin8, were used for data standardization. Expression analyses of defence-induced marker genes (Table S1) were performed by quantitative PCR with Fluidigm Biomark^® technology (Genomic GenoToul). First-strand cDNA templates were pre-amplified with TaqMan preamp master mix and reactions were achieved in a Fluidigm Biomark® BMK-M-96.96 plate according to the manufacturer’s recommendations. Relative gene expression values were determined using the 2^−∆∆CT method from Livak and Schmittgen^{59 (link)}. The expression analyses data are an average of four independent replicates. As described before, the actin8 gene expression levels were used for data standardization. In all these experiments, the wild-type plant (Col) was used as the reference.

Cheval C., Perez M., Leba L.J., Ranty B., Perochon A., Reichelt M., Mithöfer A., Robe E., Mazars C., Galaud J.P, & Aldon D. (2017). PRR2, a pseudo-response regulator, promotes salicylic acid and camalexin accumulation during plant immunity. Scientific Reports, 7, 6979.

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Quantifying gene expression in mice

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Samples from 4-month-old mice were snap-frozen in liquid nitrogen and stored at −80°C. Total RNAs were isolated using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s protocol. Purity of RNAs was assessed by a ratio of absorbance at 260 and 230 nm > 1.7. RNA quality was checked on agarose gel. One microgram of RNA was used for reverse transcription with the Maxima First Strand complementary DNA (cDNA) Synthesis Kit for reverse transcription quantitative polymerase chain reaction (RT-qPCR) (Fermentas). cDNAs were amplified using the Maxima SYBR Green qPCR Master Mix (2×; Fermentas). qPCR reactions were performed on a Roche Light Cycler system (Roche). All PCR and qPCR products were examined qualitatively on agarose gels. All presented RT-qPCR results were normalized by the geometrical mean of three independent reference genes (Ywhaz, Polr2a, and Rplpo), as previously described (49 (link)). Sequences of primers are listed in table S6.

Prola A., Blondelle J., Vandestienne A., Piquereau J., Denis R.G., Guyot S., Chauvin H., Mourier A., Maurer M., Henry C., Khadhraoui N., Gallerne C., Molinié T., Courtin G., Guillaud L., Gressette M., Solgadi A., Dumont F., Castel J., Ternacle J., Demarquoy J., Malgoyre A., Koulmann N., Derumeaux G., Giraud M.F., Joubert F., Veksler V., Luquet S., Relaix F., Tiret L, & Pilot-Storck F. (2021). Cardiolipin content controls mitochondrial coupling and energetic efficiency in muscle. Science Advances, 7(1), eabd6322.

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Gene Expression Analysis by RT-qPCR

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Total RNA was extracted from the cultured cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The RNA concentration was measured using a spectrophotometer (Thermo Fisher Scientific, Inc.) and total RNA (500 ng) was reverse transcribed into cDNA at a final volume of 10 µl using oligodT primers and SuperScript II reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using the SYBR Green reaction mix (Qiagen Corporation) and analyzed using a Roche LightCycler system (Roche Diagnostics). The following thermocycling conditions were used: Initial denaturation at 95°C for 3 min; followed by 39 cycles at 95°C for 5 sec, 56°C for 10 sec, 72°C for 25 sec; 65°C for 5 sec, and 95°C for 50 sec for the final step. The results were normalized to GAPDH expression levels and the primer sequences are listed in Table I. The RT-qPCR data were analyzed relative to the cycle threshold values and are shown as the fold change (2^−∆∆Cq) (22 (link)). The qPCR reaction was performed in triplicate for each sample.

Han Z., Meng L., Huang X., Tan J., Liu W., Chen W., Zou Y., Cai Y., Huang S., Chen A., Zhan T., Huang M., Chen X., Tian X, & Zhu Q. (2021). Inhibition of p38 MAPK increases the sensitivity of 5-fluorouracil-resistant SW480 human colon cancer cells to noscapine. Oncology Letters, 23(2), 52.

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Pancreatic Islet Gene Expression Analysis

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Pancreatic islets from WT G and Zbed6 KO G C57BL/6 mice were prepared by collagenase digestion and cultured overnight as previously described [14 (link)]. RNA was purified from islet cells using the Qiagen RNeasy kit (Qiagen, Hilden, Germany), and cDNA was synthesised using avian myeloblastosis virus (AMV) reverse transcriptase (Bio-Rad, Hercules, CA, USA). Real-time q-PCR was performed using the Roche Light Cycler System and the FastStart DNA Master DNA SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). Values were normalised to the relative amounts of GAPDH cDNA. Primer sequences can be found in electronic supplementary material (ESM Table 1).

Wang X., Younis S., Cen J., Wang Y., Krizhanovskii C., Andersson L, & Welsh N. (2021). ZBED6 counteracts high-fat diet-induced glucose intolerance by maintaining beta cell area and reducing excess mitochondrial activation. Diabetologia, 64(10), 2292-2305.

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