Taqman real‐time PCR for mRNA expression analysis was performed using the Taqman primers and probes as previously described.30 , 31 , 36 Briefly, 5 μL of suitable cDNA dilutions from unknown and standard (brain cDNA) samples and 8 μL Taqman master‐mix including primers and probes were loaded on white 384 Light cycler® 480 multiwell plates (Roche) with 18S rRNA as the internal control. Samples were loaded in triplicate, and the data were analyzed using the Light cycler® 480 system (Roche, Mannheim, Germany) as described previously.31 , 38
Light cycler 480 multiwell plates
Manufactured by Roche
The Light cycler® 480 multiwell plates are a type of lab equipment designed for use with the Light cycler® 480 real-time PCR system. The plates are made to hold and process multiple samples simultaneously during PCR (polymerase chain reaction) analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Rq1 rnase free dnase kit, by Promega (1 mentions)
Lightcycler 480 quantitative pcr system, by Roche (1 mentions)
Lightcycler 480 software v1, by Roche (1 mentions)
Allprep kit, by Qiagen (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Rna ultrasense one step quantitative rt pcr system, by Thermo Fisher Scientific (1 mentions)
5 protocols using light cycler 480 multiwell plates
1
Quantitative Real-time PCR for mRNA Expression
2
Quantifying P2X7R Expression by Real-Time PCR
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The spinal cords were homogenized in TRI reagent (Sigma-Aldrich), and the total RNA was extracted. After treatment with the RQ1 RNase-Free DNase kit to remove contaminating DNA (Promega, Madison, WI), 5 μg of total RNA was reverse transcribed for 1 hour at 50°C using 200 units of SuperScript III reverse transcriptase (Invitrogen). Then, 200 ng of cDNA was used as the template for real-time PCR.
The quantitative PCR reactions were performed in triplicate using a Roche LightCycler 480 quantitative PCR system (Roche Applied Science, Indianapolis, IN). The P2X7R expression levels were normalized to those of GAPDH. Quantitative PCR amplification was performed as 20 μL reactions containing 10 μL of X2 SYBR Green I Master Mix (Roche Applied Science), 0.8 μL of the forward and reverse primers (μM each), and 9.2 μL of the sample or nuclease-free water. All reactions were performed in 96-well plates (LightCycler 480 Multiwell Plates; Roche Applied Science).
The cycling conditions consisted of a 10-minute polymerase activation step at 95°C, 45 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 25 seconds, and a dissociation curve analysis step from 60° to 95°C. The relative quantification results for both P2X7R and GAPDH were calculated according to the second derivative maximum method using LightCycler 480 Software v.1.5 (Roche Applied Science).
The quantitative PCR reactions were performed in triplicate using a Roche LightCycler 480 quantitative PCR system (Roche Applied Science, Indianapolis, IN). The P2X7R expression levels were normalized to those of GAPDH. Quantitative PCR amplification was performed as 20 μL reactions containing 10 μL of X2 SYBR Green I Master Mix (Roche Applied Science), 0.8 μL of the forward and reverse primers (μM each), and 9.2 μL of the sample or nuclease-free water. All reactions were performed in 96-well plates (LightCycler 480 Multiwell Plates; Roche Applied Science).
The cycling conditions consisted of a 10-minute polymerase activation step at 95°C, 45 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 25 seconds, and a dissociation curve analysis step from 60° to 95°C. The relative quantification results for both P2X7R and GAPDH were calculated according to the second derivative maximum method using LightCycler 480 Software v.1.5 (Roche Applied Science).
3
RNA Extraction and qRT-PCR Analysis
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Cellular RNA of 2-5x105 cells was extracted using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. For lysis, 1% 2-mercaptoethanol was added to the lysis buffer. 200 ng of extracted cellular RNA was used for cDNA transcription using the QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer’s instructions. cDNA samples were filled up to 60 µl with nuclease-free water. qRT-PCR measurements were carried out on a Lightcycler 480 (Roche) using Lightcycler 480 multiwell plates (Roche) and Luna Universial qPCR Master Mix (NEB) according to the manual. In brief, a master mix containing primers (final concentration 0.3 µM), Luna Universial qPCR Master Mix and nuclease free water was generated and added to the wells together with 2 µl of diluted cDNA in duplicates (Table 4
4
Quantitative PCR and Western Blot Analysis
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Tissues were processed with the AllPrep kit (Qiagen, Hilden, DE) per manufacturer guidelines and RNA stored until use. In vitro cell culture RNA was isolated using the Qiagen RNeasy Mini Kit, and protein was precipitated using buffer APP (Qiagen) per manufacturer’s guidelines). RNA was measured using a NanoDrop™ spectrophotometer (Wilmington, USA). Reverse transcription was performed using the QuantiTect® Reverse Transcription Kit (Qiagen) with Fast Start Essential DNA Green Master (Roche). cDNA, SYBR Green and ultrapure dH2O were combined and loaded on to LightCycler® 480 Multiwell plates (Roche) and run on a LightCycler® 480 Instrument II (Roche); qPCR primers are included in Additional file 1 : Table S2. Data was subsequently analyzed using the LightCycler® 480 software (Version 1.5, Roche). Standard curves (Arnt2, Npas4, Bdnf, and β-actin cDNA generated from mouse brain, diluted in 10-fold successions) were included on each run for absolute quantification. Precipitated proteins from spinal cord were quantified and analyzed by western blotting as outlined above for cell cultures.
5
Quantitative RT-qPCR for Nucleic Acid Quantification
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RT-qPCR was performed as recommended by ISO 15216-1, with an RNA UltraSense™ One-Step Quantitative RT-PCR System (Thermo Fisher Scientific) in LightCycler® 480 Multiwell Plates on a LightCycler® 96 System (both Roche, Basel, Switzerland). Each reaction contained 500 nM forward primer, 900 nM reverse primer and 250 nM probe. For each sample, a total of 25 µL reaction mix was prepared, with 20 µL of reagent and 5 µL of sample. RT-qPCR was run with RT at 55 °C for 1 h, followed by inactivation of the reverse transcriptase at 95 °C for 5 min. There were 45 cycles, with denaturation of the template at 95 °C for 15 s, annealing at 60 °C for 1 min and elongation at 65 °C for 1 min. The results were analysed with the LightCycler® 96 software, version 1.1 (Roche). The Cq values were determined by the software. Quantification by RT-qPCR was performed using a tenfold dilution series of linearised plasmid DNA, ranging from 105 to 10 copies/reaction (Table 1 ). Only one standard (Table 1 ) was used for quantification of the different primer combinations, as it became apparent that the different primer systems were similar in their performance (see section “Primers and Probes ”).
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