Total RNA was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocols. The quantity of total RNA was measured by spectrophotometry. Complementary DNA was synthesized using a First Strand cDNA Synthesis Kit (TAKARA, Japan) according to the manufacturer's instructions. Real-time polymerase chain reaction (RT-PCR) was performed on a 96-well plate ABI Prism 7500 (Applied Biosystems, Foster City, CA) using a KAPA SYBR FAST qPCR Kit Master Mix. Relative expression was calculated using the 2−ΔΔCt method normalized to β-actin.
Kapa sybr fast qpcr kit master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The KAPA SYBR FAST qPCR Kit Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains all the necessary components, including a DNA polymerase, buffer, and dye, to perform qPCR reactions.
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Lab products found in correlation
3 protocols using kapa sybr fast qpcr kit master mix
1
RNA Extraction and Real-Time PCR
2
Wheat Gene Expression Quantification
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One μg of total RNA was used for cDNA synthesis using the SuperScript® III kit (Thermo Fisher Scientific) as per manufacturers’ instructions. Quantitative real–time PCR was performed with KAPA SYBR® Fast qPCR kit Master Mix, and amplification was real–time monitored on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Gene-specific primers targeted to amplify all three homeologs simultaneously were designed (Supplementary Table 1 ) and the specificity of each pair was verified by melt curve analysis and sequencing of the products. Change in gene expression (calibrated normalized relative quantities, CNRQ) were calculated using qBASE+ software (Hellemans et al., 2007 (link)). Specifically, gene expression was normalized to wheat reference genes (cyclophilin and elongation factor alpha) and then scaled to the 1 day +N weighted mean.
3
Quantitative Real-Time PCR for Gene Expression
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Quantitative real-time PCR was performed with KAPA SYBR ® Fast qPCR kit Master Mix, and amplification was real-time monitored on a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, USA). Gene-specific primers targeted to amplify all three homeologs simultaneously were designed with AlleleID® software (Premiere Biosoft) (Table S3) and the specificity of each pair was verified by melting curve analysis and sequencing of the products. Change in gene expression were calculated using qBASE + software and reported as calibrated normalised relative quantities (CNRQ) that represents the relative gene expression level between different samples for a given target gene:
NRQ is then divided by a calibration factor (CF) (Hellemans et al. 2007 ). Four reference genes (Table S3) were quantified by qRT-PCR: TaActin, TaGAPdH, TaCyclophilin and TaEFA2. CNRQ values were calculated using the most stable genes within a specific tissue (selected by qBASE + software).
NRQ is then divided by a calibration factor (CF) (Hellemans et al. 2007 ). Four reference genes (Table S3) were quantified by qRT-PCR: TaActin, TaGAPdH, TaCyclophilin and TaEFA2. CNRQ values were calculated using the most stable genes within a specific tissue (selected by qBASE + software).
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