Cultured cells were harvested using TRIzol reagent (Sigma, USA), and the mRNA was extracted from the TRIzol reagent using an RNA extraction kit (Invitrogen, USA) according to the manufacturer's instructions. We used reverse transcription reagents and real‐time PCR mix (Vazyme Biotech, China) on a LightCycler® 96 (Roche Life Sciences). The expression levels of all genes (Table 1 ) were determined relative to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene expression. qPCRs were performed in triplicate from two independent experiments.
Real time pcr mix
Manufactured by Roche
Sourced in China, Switzerland, United States
The Real-time PCR mix is a ready-to-use solution designed for real-time polymerase chain reaction (PCR) analysis. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to amplify and detect target DNA sequences in a single reaction.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 real time pcr system, by Roche (3 mentions)
First strand synthesis kit, by Roche (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Rna extraction kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Lightcycler 96, by Roche (1 mentions)
M mlv reverse transcriptase, by Takara Bio (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
Reverse transcription reagent, by Vazyme (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lightcycler, by Roche (1 mentions)
5 protocols using real time pcr mix
1
Quantitative real-time PCR analysis
2
Quantifying mRNA Expression Levels
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To quantify the expression levels of mRNA, root tips from 5-day-old seedlings were harvested after being transferred to 22 °C and 30 °C for 24 h. Total RNA was extracted by hot phenol method and then treated with RNase-free DNase I (Takara, Shiga, Japan) to remove genomic DNA. Complementary DNAs were synthesized from total RNA primed with oligo(dT)18 primers using M-MLV reverse transcriptase (Takara) according to the user’s manual. Semi-quantitative PCR for cals8-1, cals8-2, and cals6 was performed using specific primers across the T-DNA insertion site and eIF-4A as an internal standard. Quantitative PCRs were performed using gene-specific primers and real-time PCR mix (Roche, Basel, Switzerland) in a LightCycler 480 Real-Time PCR System (Roche) with a standard program for 40 cycles. The levels of gene expression were calculated relative to eIF-4A. The primer sequences are listed in Supplementary Table S1 .
3
Quantification of Arabidopsis plm-2 mRNA
4
Quantification of Arabidopsis plm-2 mRNA
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For quantification of mRNA, root tips from 5-day-old seedlings were harvested. The total RNA was extracted with a miRNeasy Mini Kit (QIAGEN) and treated with DNase on a column. cDNAs were synthesized from total RNA primed with oligo(dT)18 primers using a First strand synthesis kit (Roche). Semi-quantitative PCR for plm-2 was performed using specific primers across the T-DNA insertion site and ACTIN2 as an internal standard. Quantitative PCRs (qPCRs) were performed using gene-specific primers and real-time PCR mix (Roche) in a LightCycler 480 Real-Time PCR System (Roche) with a standard program for 40 cycles. The levels of expression were calculated relative to ACTIN2. Primer sequences are provided in Supplemental Table 1 .
5
Quantification of OPN mRNA Expression
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Total RNA was isolated from cell pellets (1 ×106 per well in 12 wells plate) using TRIzol reagent (Life Technologies, Grand Island, NY, USA), cDNA was made from RNA samples (1000 ng per sample) using reverse transcription reagents (Vazyme Biotech Co. Ltd, Nanjing, China) and quantitative PCR assays were carried out to quantify mRNA expression levels of OPN, using a Real-Time PCR Mix (Vazyme azymeime PCR Mix in a Light Cycler (Roche Molecular Biochemicals, Indianapolis, IN, USA), with GAPDH gene used as the internal loading control. The following primer sequences were used: OPN (forward primer: 5′-GTGGGAAGGACAGTTATCAA-3′; reverse primer: 5′-CTGACTTTGGAAAGTTCCTG-3′); GAPDH (forward primer: 5′-CAATGACCCCTTCATTGACC -3; reverse primer, 5′-GACAAGCTTCCCGTTCTCAG -3′).
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