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Lightcycler 96 real time pcr system

Manufactured by Roche
Sourced in Switzerland, United States, Germany, China, Japan, United Kingdom, Poland, Denmark, Canada, France

The LightCycler 96 Real-Time PCR System is a compact and versatile real-time PCR instrument designed for accurate and reliable nucleic acid quantification. It features a 96-well microplate format and is capable of performing real-time PCR analyses for a wide range of applications.

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660 protocols using lightcycler 96 real time pcr system

1

Quantification of Influenza D and Mycoplasma bovis

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Influenza D virus was quantified in nasal swab, BAL fluid, and tissue samples using a one-step RT-qPCR as previously described (10 (link)). Briefly, the viral polymerase basic 1 (PB1) gene was amplified with specific primers and quantified by using a specific probe and the QuantiNova probe RT-PCR kit (Qiagen, Germany) on a LightCycler 96 real-time PCR system (Roche, Switzerland). Viral copy numbers in samples were determined by using a standard plasmid containing the PB1 product of influenza virus D/bovine/France/5920/2014 (18 (link)). For quantification of M. bovis genomic DNA copy numbers in nasal swab, BAL fluid, and tissue samples, qPCR was performed with specific primers, probe, and specific standard using the Bio-T Mycoplasma bovis PCR kit (Biosellal, France) on the LightCycler 96 real-time PCR system (Roche, Switzerland) according to the manufacturer’s instructions.
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2

Quantification of Influenza D and Mycoplasma bovis

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Influenza D virus was quantified in nasal swab, BAL fluid, and tissue samples using a one-step RT-qPCR as previously described (10 (link)). Briefly, the viral polymerase basic 1 (PB1) gene was amplified with specific primers and quantified by using a specific probe and the QuantiNova probe RT-PCR kit (Qiagen, Germany) on a LightCycler 96 real-time PCR system (Roche, Switzerland). Viral copy numbers in samples were determined by using a standard plasmid containing the PB1 product of influenza virus D/bovine/France/5920/2014 (18 (link)). For quantification of M. bovis genomic DNA copy numbers in nasal swab, BAL fluid, and tissue samples, qPCR was performed with specific primers, probe, and specific standard using the Bio-T Mycoplasma bovis PCR kit (Biosellal, France) on the LightCycler 96 real-time PCR system (Roche, Switzerland) according to the manufacturer’s instructions.
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3

RNA Isolation and qRT-PCR Analysis of lncRNA, circRNA, and miRNA

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For lncRNA and circRNA, total RNA isolation and cDNA preparation from different tissue samples of GRS and HG were the same as those above in “RNA isolation and quality control.” The quantitative real-time PCR (qRT-PCR) analysis was performed using 2 × RealStar Green Fast Mixture (GenStar, China) and the Real-Time PCR System (Lightcycler 96, Roche, Switzerland). β-actin was used as an internal control gene. For each reaction, 0.5 μl of the forward and reverse primers and 2 μl of cDNA template were added.
In PCR validation of miRNA expression, total RNA reverse transcription was performed using the Mir-X miRNA First-Strand Synthesis kit (TaKaRa, Dalian, China). The 5′ forward primers for qRT-PCR validation of miRNAs included the entire sequence of the mature miRNAs, as suggested by the manufacturer, and the 3′primer for qRT-PCR was supplied with the kit. The qRT-PCR analysis was performed using TB Green Premix Ex Taq II (TaKaRa, Dalian, China) and the Real-Time PCR System (Lightcycler 96, Roche, Switzerland). U6 was used as the internal control. For each reaction, 0.8 μl of the forward and reverse primers and 2 μl of cDNA template were added.
All of the primers used in this study are listed in Table S13. The relative gene expression level was calculated according to the 2−ΔΔCt method56 .
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4

Quantitative RT-PCR Gene Expression Analysis

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Conversion of total RNA to cDNA and qPCR was performed with GoTaq 2 -Step RT-qPCR System (Promega Corp, catalog #A6010, Madison, WI) using primers listed in Table S2 according to manufacturer’s instructions. RT-qPCR was performed with a LightCycler 96 Roche Real-Time PCR system (Roche Diagnostics, Indianapolis, IN). Briefly, 88 ng of cDNA was amplified by qPCR with 1 µM of each primer using the following conditions: preincubation (95C for 60S), followed by 45 amplification cycles (95C for 10 s, 55C for 10 s, 72C for 10 s), and a final cycle (95C for 5 s, 72C for 30 s, and cooling at 37C for 30 s). QPCR reactions were performed in duplicate and all genes listed in Table S2 were analyzed at the same time. Each 96 well plate included one control and one infected sample. Relative gene expression for each specimen was determined by the comparative Cq method with Actb used as the reference and values reported as 2−(ΔCq)40 (link). All primer pairs listed in Table S2 have similar primer efficiencies as shown by logarithmic PCR amplification plots40 (link) (Fig. S1).
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5

Real-time PCR Gene Expression Analysis

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Real-time PCR was performed using the LightCycler 96 Roche Real-time PCR system and PowerUp™ SYBR™ Green Master Mix (applied biosystem by Thermo Fisher Scientific). Seventeen differentially expressed genes (from shoots and roots of both low N and high N) were selected for validation. Primer3 version 2.4.0 was used to design gene-specific primers and their specificity was verified using the NCBI database through the Blast tool (Supplementary Table S5). The 10 µl RT-qPCR reaction contained 1 µl of template cDNA (20 ng), 1 µl of forward primer, 1 µl of reverse primer, 4 µl of PowerUp™ SYBR™ Green Master Mix, and 3 µl of H2O. PCR was run at an initial denaturation of 94°C for 3 min followed by 40 cycles of 94°C for 10 s, 60°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min to check the specificity of amplification. The housekeeping gene TaATP (ATP-dependent 26S proteasome regulatory) (Paolacci et al., 2009 (link)) was used as the endogenous control and all reactions were performed in triplicate. Relative gene expression was analyzed using the 2−ΔΔCt method.
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6

RNA Extraction and qPCR Analysis of Uterine Tissues

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At time of necropsy, the mesometrial triangle and decidua was removed and excess uterine tissue was trimmed away from the mesometrial triangle before immersion in Trizol Reagent (Life Technologies, Cat# 15596-018) at a ratio of 1 mL of reagent per 50–100 mg of tissue. Samples were stored at − 80 °C in RNAase free tubes until processing. RNA extraction and assessment of RNA quality was performed as previously described11 (link). Total RNA was converted to cDNA and qPCR was performed with GoTaq 2-Step RT-qPCR System (Promega Corp, catalog #A6010, Madison, WI) using primers listed in Table S2 according to manufacturer’s instructions. RT-qPCR were performed as previously described11 (link). All primer pairs listed in Table S3 have similar primer efficiencies determined by logarithmic PCR amplification plots11 (link),36 (link). RT-qPCR reactions were performed with a LightCycler 96 Roche Real-Time PCR system (Roche Diagnostics, Indianapolis, IN) using the same amplification conditions as already described11 (link).
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7

Gallbladder Tissue Analysis of ZHX1 Expression

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The gallbladder tissues used for determination of ZHX1 expression were provided by the Biobank of Pusan National University Hospital, a member of the Korea Biobank Network. The tissues were obtained with informed consent from patients who underwent surgical resection at Pusan National University Hospital and the present study was approved by the institutional review board of the hospital (PNUH-IRB). Total RNAs were extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). Purified RNA were used to synthesize cDNA using oligo-dT, dNTP, RNasin, MMLV reverse transcriptase (Promega, Madison, WI, USA). cDNAs obtained were used as templates for real-time PCR. cDNAs were mixed with FastStart Essential DNA Green Master (Roche Diagnostics GmbH, Mannheim, Germany) to determine ZHX1 mRNA levels. Real-time PCR and analysis were performed on a LightCyclerTM 96 Real-time PCR system (Roche, Nonnenwald, Germany). Three separate experiments were performed and GAPDH was used as the internal control. The primer sequences for real-time PCR were as follows: GAPDH forward primer 5’- TGG TGA CCA GGC GCC CAA TAC G -3’, GAPDH reverse primer 5’- GCA GCC TCC CGC TTC GCT CT -3’, ZHX1 forward primer 5’- TCC CTT ACC CAA CAA TGT CA- 3’, ZHX1 reverse primer 5’- TTG TTT CCT TCT TGC CTC CT -3’, EGR1 forward primer 5’- CTT TTC CCT GGA GCC TGC AC -3’, EGR1 reverse primer 5’- AAT GTC AGT GTT CGG CGT GG -3’.
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8

Quantitative Real-Time PCR for Gene Expression

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Total RNAs were extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from purified RNA using oligo-dT, dNTP, RNasin, and MMLV reverse transcriptase (Promega). The cDNAs obtained were used as template for real-time polymerase chain reaction (PCR). The prepared cDNA, specific primers, and FastStart Essential DNA Green Master (Roche, Nonnenwald, Germany) were mixed together to determine relative ZHX1 mRNA expression. Real-time PCRs were performed on a LightCycler TM 96 Real-Time PCR system (Roche). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. Three separate experiments were performed. The primer sequences for real-time PCR were as follows: GAPDH, 5′-TGG TGA CCA GGC GCC CAA TAC G-3′ and 5′-GCA GCC TCC CGC TTC GCT CT-3′; ZHX1, 5′-TCC CTT ACC CAA CAA TGT CA-3′ and 5′-TTG TTT CCT TCT TGC CTC CT-3′; SNAI1, 5′-GAG GCG GTG GCA GAC TAG-3′ and 5′-GAC ACA TCG GTC AGA CCA G -3′; SNAI2, 5′-TAG GAA GAG ATC TGC CAG AC-3′ and 5′-CCC CAA GGC ACA TAC TGT TA-3′; TWIST1, 5′-CGG GAG TCC GCA GTC TTA-3′ and 5′-TGG ATC TTG CTC AGC TTG TC-3′; TWIST2, 5′-CTT ATG TTT GGG GGG AGG TT-3′ and 5′-TAG CCA AGC AAT CAC GGA GA-3′.
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9

Relative-real-time PCR analysis of azurin gene expression in P. aeruginosa

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Relative-real-time PCR was performed to determine the expression level of azurin gene in different isolates of P. aeruginosa using the Light Cycler 96 Real-Time PCR system (Roche Life Science, Germany). Each PCR reaction was performed in a total reaction volume of 20 μL containing 12 μL of real-time PCR Master Mix (Amplliqon, Denmark), 1 μL cDNA template, 1 μL of each primer (Blu), and 5 μL distilled water. Primer designation and sequences are depicted in Table 2. The qPCR was performed according to the following conditions: pre-incubation at 95°C for 5 min, 45 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 30 s. The analysis of melting pick was performed at 95°C for 5 min. In each sample, the same amount of RNA was used, which were converted into the cDNA and pipetted. Ribosomal protein S12 (Rpsl) mRNA expression was applied as the internal control for each sample (Dumas et al., 2006), and the ΔCT (CT target − CT reference) and expression fold change were calculated for each sample according to the comparative CT method (Pfaffl formula).25 (link) Each real-time PCR reaction was performed in duplicate, and the standard deviation was calculated. The efficiency of real-time PCR was determined by amplifying a serial dilution of the template cDNA (10 folds) and calculating E= −1+10 (−1/slope).
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10

Quantifying plant gene expression

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Total RNA was extracted from 4-week-old plants using an RNeasy kit (Qiagen, Germany), and 2 µg of RNA was reverse-transcribed using SuperScript IV Reverse Transcriptase according to the manufacturer's instructions (Thermo Fisher Scientific). Real-time PCR was performed on complementary DNAs in a final volume of 10 µL using SYBR (Synergy Brands, Inc) Green I master mix (Roche Life Science) and specific primers: for CLCa, 5′-ATCAAATGGAGATGGCTTCG-3′ (Forward) and 5′-CCTCAAGAGCGAAAAGTACTC-3′ (Reverse); and for the ACTIN2 reference gene, 5′-GGTAACATTGTGCTCAGTGGTGG-3′ (Forward) and 5′-AACGACCTTAATCTTCATGCT-3′ (Reverse). The reactions were performed in a LightCycler 96 real-time PCR system (Roche Life Science). Samples were subjected to ten minutes of pre-incubation at 95°C, followed by 45 amplification cycles of 15 s at 95°C, 15 s at 60°C, and 15 s at 72°C. High-resolution melting was performed to assess amplification specificity, and several cDNA dilutions were tested to calculate primer efficiency. The results were analyzed using LightCycler Software (Roche Life Science) and normalized to ACTIN2 gene expression.
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