Tissue was collected 3 weeks after TRV inoculation to test the down‐regulation of NbRPL10 and other ribosomal protein‐encoding gene transcripts in N. benthamiana‐silenced plants. Leaf tissue was collected 3 weeks after germination to determine the expression of AtRPL10A, AtRPL10B, and AtRPL10C in Arabidopsis wild type (Col‐0), AtRPL10A overexpression, and RNAi or mutant plants. Total RNA was extracted according to the manufacturer's instructions using a Qiagen total RNA extraction kit. The cDNA was synthesized by oligo (dT) primers using Molony murine leukaemia virus reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer's instructions. The real‐time RT‐qPCR was performed with a Sigma KicQStart SYBR green kit. The conditions for PCR were as follows: 95°C for 2 min, 25 cycles of denaturation at 94°C for 45 s, annealing for 30 s at 58°C, polymerization for 45 s (72°C), followed by plate reading at 72°C for 5 min, estimation of melting curve from 50°C to 95°C, and incubation at 72°C for 4 min. The primers used in the study are given in Table S1 .
Kicqstart sybr green kit
Manufactured by Merck Group
Sourced in United States
The KicQStart SYBR green kit is a laboratory reagent used for quantitative real-time PCR (qPCR) applications. The kit contains SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence. This allows for the detection and quantification of DNA sequences during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Total rna extraction kit, by Qiagen (1 mentions)
Thermoscript rt pcr system for first strand cdna synthesis, by Thermo Fisher Scientific (1 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirvana kit, by Thermo Fisher Scientific (1 mentions)
Kicqstart sybr green predesigned primers, by Merck Group (1 mentions)
Lightcycler 480, by Roche (1 mentions)
3 protocols using kicqstart sybr green kit
1
Quantifying Ribosomal Protein Gene Expression
2
Quantitative Analysis of miR-34a and Gene Expression
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Total RNA was isolated from cell lines using the mirVana kit (Ambion, Austin, TX) according to the manufacturer's instructions and 10 ng of total RNA was reverse transcribed using the TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative PCR (qPCR) was performed on the Agilent 3000P system using the human miR-34a and U6 miRNA TaqMan expression assays (Applied Biosystems). Relative miR-34a expression was determined using the gene comparative CT method. For gene expression analysis, 200 ng of total RNA was reverse transcribed using ThermoScriptTM RT-PCR system for first strand cDNA synthesis (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Gene expression was then determined by qPCR using the KiCq Start SYBR Green kit (Sigma, St. Louis, MO). The primers sequences used for gene expression SYBR Green qPCR are listed in Supplemental Table 1 .
3
Quantitative PCR analysis of stem cell markers
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The quantitative PCR reaction was performed for CD133, CD166, EpCAM and TACSTD2 using the KiCqStart SYBR Green kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. KiCqStart SYBR Green predesigned primers (Sigma, St. Louis, MO, USA) were employed. All primers were used in a final concentration of 300 nM, and 10 ng of cDNA were used per well, for a total volume of 10 µL. All cDNA samples were measured in triplicate in a 96-well plate covered with adhesive seals in the thermocycler Roche LightCycler 480 (Roche, Basel, Switzerland). Reactions started with 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 1 min at 60 °C and 10 s at 72 °C. The 2−ΔCT method was used for calculating the normalized mRNA expression. Beta-actin was used as a housekeeping gene to normalize samples.
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