RNA was extracted using the Tri-Reagent protocol (T9424, Sigma-Aldrich) and 1µg was reverse transcribed with the High Capacity cDNA reverse transcription kit (44368813, Thermo Fisher Scientific) using random hexamer primers. The PCR reaction containing SensiMix™ SYBR Green® No-ROX Kit (QT650-20, Bioline, London, UK) was run on a 7900 Real time PCR System (Applied Biosystems, Foster City, California) with standard cycling conditions: 10 minutes 95°C, and 40 cycles of 15 seconds 95°C followed by 1 minute 60°C. Gene expression was analysed with the Ct method using HPRT1 expression for normalization. The primers used are listed in Supplementary Table 3
Sensimix sybr green no rox kit
Manufactured by Meridian Bioscience
Sourced in United Kingdom
The SensiMix™ SYBR Green® No-ROX Kit is a ready-to-use solution for quantitative real-time PCR (qPCR) assays. It contains all the necessary components, including the SYBR Green dye, required for sensitive and accurate gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tri reagent, by Merck Group (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Forward and reverse primer, by Merck Group (1 mentions)
Epmotion 5075 robot, by Eppendorf (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
4 protocols using sensimix sybr green no rox kit
1
Quantitative RT-PCR Gene Expression Analysis
2
Quantifying Gene Expression by RT-qPCR
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RNA was extracted using the Tri-Reagent protocol (T9424, Sigma) and 1 μg of RNA was reverse transcribed into cDNA with the High Capacity cDNA reverse transcription kit (44368813, Thermo Fisher) using random hexamer primers. The PCR reaction containing SensiMix™ SYBR Green® No-ROX Kit (QT650–20, Bioline) was run on a 7900 Real time PCR System with standard cycling conditions: 10 min 95 °C, and 40 cycles of 15 s 95 °C followed by 1 min 60 °C. Gene expression was analysed with the Ct method using HPRT1 expression for normalization [28 ]. The primers used are listed in Supplementary Table 2 .
3
RNA Extraction and qRT-PCR Analysis
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RNA was extracted using the Tri-Reagent protocol (T9424, Sigma) and 1µg of RNA was reverse transcribed into cDNA with the High Capacity cDNA reverse transcription kit (44368813, Thermo Fisher) using random hexamer primers. The PCR reaction containing SensiMix™ SYBR Green → No-ROX Kit (QT650-20, Bioline) was run on a 7900 Real time PCR System with standard cycling conditions: 10 min 95°C, and 40 cycles of 15s 95°C followed by 1 min 60°C. Gene expression was analysed with the Ct method using HPRT1 expression for normalization 26 . The primers used are listed in Supplementary table 2.
4
Transcriptional Analysis of Sorghum Nitrogen Metabolism
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Transcript levels of CYP79A1 (Sb01g001200), NIT4B2 (Sb04 g026940), nitrate reductase 1 (NR1, Sb07g022750) and glutamine synthetase 2 (GS2, Sb06g031460) normalised to ubiquitin (UBT, Sb01g030340) (Paolacci et al. 2009) (link) were determined by qPCR using a Light Cycler 480II (Roche) for three replicates of both the leaves and roots of each line (Sb, acdc1 and tcd1), nitrogen level (LN, MN and HN) and leaf stage (three-, five-, eight-and 10-leaf) (Fig. 1b). The sorghum genome has been sequenced (Paterson et al. 2009 (link)) providing a resource to identify the sequences of the selected genes. Forward and reverse primers (Sigma-Aldrich), specific for a region in the 3 0 end of each gene transcript sequence, were designed using PerlPrimer (http://perlprimer.sourceforge.net/; see Table S1, available as Supplementary Material to this paper). The Epmotion 5075 Robot (Eppendorf) was used to set up the 384 well plates. SensiMix SYBR Green No ROX kit (Bioline) was used for qPCR according to the manufacturer's instructions. Each sample was run in triplicate along with a set of standards specific for the gene being analysed. Each RNA preparation (i.e. the RNA used to create the cDNA) was also checked for DNA contamination by using crude RNA as the template and determining the occurrence of any amplification (data not shown).
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