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.
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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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (138 mentions)
Trizol, by Thermo Fisher Scientific (37 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (30 mentions)
Sybr premix ex taq 2, by Takara Bio (23 mentions)
Primescript rt reagent kit, by Takara Bio (21 mentions)
Primescript rt master mix, by Takara Bio (21 mentions)
Faststart essential dna green master, by Roche (20 mentions)
Lightcycler 96, by Roche (20 mentions)
Rneasy mini kit, by Qiagen (19 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (17 mentions)
Tb green premix ex taq 2, by Takara Bio (16 mentions)
Trizol reagent, by Takara Bio (15 mentions)
Quantitect reverse transcription kit, by Qiagen (13 mentions)
Rneasy kit, by Qiagen (13 mentions)
Faststart universal sybr green master, by Roche (11 mentions)
Sybr premix ex taq, by Takara Bio (11 mentions)
Gotaq qpcr master mix, by Promega (10 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (10 mentions)
Nanodrop, by Thermo Fisher Scientific (10 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (10 mentions)
658 protocols using lightcycler 96 real time pcr system
1
Quantification of Influenza D and Mycoplasma bovis
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.
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 TableS13 . The relative gene expression level was calculated according to the 2−ΔΔCt method56 .
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
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 ).
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.
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).
7
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).
8
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.
9
Quantitative Real-Time PCR Protocol
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The seven small-molecule compounds were purchased from Topscience (China); the CAS ID and product ID are in Supplementary Table 3 . MEFs were treated with chemicals for 7 days, and total RNA was isolated using the RNeasy Micro Kit (QIAGEN). RNA was converted to cDNA using First-Strand Synthesis SuperMix for quantitative real-time PCR (qRT-PCR) (INVITROGEN). PCR was carried out using Power SYBR Green PCR Kit (Applied Biosystems, Foster City, CA) and a LightCycler 96 Real-Time PCR System (Roche, Mannheim, Germany). The data were analyzed using the 2−ΔΔCt method. The primers were listed in Supplementary Table 9 .
10
Quantitative Real-time PCR Analysis of ABCA1, COL6A1, and GSTT1L
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Quantitative Real-time PCR (Q-PCR) was used to measure the mRNA expression levels of the ABCA1, COL6A1, and GSTT1L genes. Total RNA was extracted from the breast muscle tissues using the TRIzol reagent (Invitrogen) and was further purified with RNeasy columns (Qiagen) according to the manufacturer’s protocol. The primers for the Q-PCR were designed using online software Primer3plus82 (link) (http://primer3.sourceforge.net/webif.php ) and were synthesized by Sangon biotech Co., Ltd. (Shanghai, China) (Supplementary Table S5 ). Each of the RNA samples was diluted to 1000 ng/uL. The cDNA was synthesized using the primeScriptTM RT reagent kit (Takara). Q-PCR was performed using SYBR® Premix Ex TaqII (Takara) on the LightCycler® 96 Real-Time PCR system (Roche Applied Science) in a 10-uL reaction volume containing 1 μL cDNA, 5 μL 2× SYBR®Premix Ex Taq™ II (TliRNaseH Plus) (TaKaRa), 0.5 μL each of forward and reverse primers (10 μM), and 3 μL RNase-Free Water. All of the data were normalized to the expression level of actin. The Q-PCR amplification procedure was as follows: 95 °C for 3 min; 35 cycles of 95 °C for 30 s; 60 °C for 30 s; 72 °C for 20 s; and an extension for 10 min at 72 °C. All of the Q-PCR reactions were performed in triplicate. The 2−ΔΔCt method was used to determine the relative mRNA abundance83 (link).
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