The total RNA extraction was done using a TRIzol reagent (Invitrogen). The concentration of RNA was quantified by utilizing a NanoDrop spectrophotometer. Complementary DNA (cDNA) was synthesized using a Reverse Transcription Supermix Kit (Bio-Rad, 1708841) according to the manufacturer’s protocol. The qRT-PCR reaction was carried out on a Mastercycler RealPlex2 real-time PCR system (Eppendorf) using the Power-up SYBR Green Master Mix (Applied Biosystems, A25741) with gene-specific primers. Relative mRNA expression was calculated by normalizing to GAPDH mRNA levels.
Mastercycler realplex2 real time pcr system
Manufactured by Eppendorf
Sourced in Belgium
The Mastercycler Realplex2 is a real-time PCR system designed for quantitative analysis of DNA samples. It features a 96-well thermal block and provides accurate temperature control. The system is capable of performing real-time PCR reactions and collecting fluorescence data during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Takyon no rox sybr master mix blue dttp, by Eurogentec (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Reverse transcription supermix kit, by Bio-Rad (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Avian myeloblastosis virus reverse transcriptase xl, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green, by Qiagen (1 mentions)
Omniscript rt, by Qiagen (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna plus kit, by Macherey-Nagel (1 mentions)
5 protocols using mastercycler realplex2 real time pcr system
1
Total RNA Extraction and qRT-PCR Analysis
2
RNA Extraction and qRT-PCR Analysis
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Total RNA from root tissues of 30 seedlings was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. DNA-free total RNA (2 µg) from different treatments was used for first-strand cDNA synthesis in a 20-µL reaction volume containing 2.5 units of avian myeloblastosis virus reverse transcriptase XL (TaKaRa) and oligo dT primer.
Real-time quantitative RT-PCR reactions were performed with Mastercycler realplex2 real-time PCR system (Eppendorf, Hamburg, Germany) using the SYBR Premix Ex Taq (TaKaRa) according to the user manual. The cDNA was amplified using primers (Table S1 ). The expression levels of the genes are presented as values relative to the corresponding control samples under the indicated conditions, with normalization of data to the geometic average of two internal control genes MSC27 and Actin2[46] (link).
Real-time quantitative RT-PCR reactions were performed with Mastercycler realplex2 real-time PCR system (Eppendorf, Hamburg, Germany) using the SYBR Premix Ex Taq (TaKaRa) according to the user manual. The cDNA was amplified using primers (
3
Quantitative PCR Protocol for Gene Expression Analysis
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The qPCRs were performed in triplicate using Takyon No ROX SYBR MasterMix blue dTTP (Eurogentec, Liège, Belgium) and a Mastercycler Realplex2 Real-Time PCR System (Eppendorf, Hamburg, Germany). The cycling program was: 5 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 45 s at the annealing temperature indicated in Table 1 . Relative expression of Cyclin D1 transcripts was standardized using 3 housekeeping transcripts (EIF3F, RPL13A and PPIA) using a method previously described [22 (link)]. Primers used to amplify transcripts were designed in different exons to avoid the amplification of potential genomic DNA traces. Primer specificity was checked using a Basic Local Alignment Search Tool (BLAST) search through the US National Center for Biotechnology Information (Bethesda, MD, USA).
4
Quantitative PCR Analysis of RNA Expression
5
Quantitative RT-PCR Analysis of Splenic DCs and BMDCs
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Total RNA was extracted from splenic DCs or BMDCs using the NucleoSpin®RNA Plus kit (Macherey-Nagel, Düren, Germany) and reverse transcribed using random primers, dNTP, RNAseout, DTT, and MML-V reverse transcriptase (all from Invitrogen, Cergy Pontoise, France), following the manufacturer’s instructions.
qPCRs were performed in triplicate using Takyon No ROX SYBR MasterMix blue DTTP (Eurogentec, Liège, Belgium) and a Mastercycler Realplex2 Real-Time PCR System (Eppendorf, Hamburg, Germany). The cycling program was 5 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 45 s at the annealing temperature indicated inTable 1 . The relative expression of transcripts of interest was standardized using 2 housekeeping transcripts (Gusb/Eif3f for BMDCs and Gusb/Ef1a for splenic DCs) using a method previously described [32 (link)]. Primers (Eurogentec, Liège, Belgium) used to amplify transcripts were designed in different exons to avoid the amplification of potential genomic DNA traces. Primer specificity was checked using a Basic Local Alignment Search Tool (BLAST) search through the US National Center for Biotechnology Information (Bethesda, MD, USA).
qPCRs were performed in triplicate using Takyon No ROX SYBR MasterMix blue DTTP (Eurogentec, Liège, Belgium) and a Mastercycler Realplex2 Real-Time PCR System (Eppendorf, Hamburg, Germany). The cycling program was 5 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 45 s at the annealing temperature indicated in
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