Total RNA was extracted by TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. 1 μg of total RNA was reverse-transcribed by the reverse transcription kit (Promega, Madison, WI, USA) and PCR was performed using Cycler Apparatus (Bio-Rad, Hercules, CA, USA). For PCR amplification, the following primers were used: GAPDH (580 bp), 5'-AAGCCCATCACCATCTTCCA-3' (Forward) and 5'-CCTGCTTCACCACCTTCTTG-3' (Reverse); Mipu1 (1800 bp), 5'-ATGCCTGCAGCCCGAGGGAAATC-3' (Forward) and 5'-CTTAGGACATTTCCTCCGAATG-3' (Reverse). The PCR reaction consisted of 26 cycles of denaturing at 95 °C for 30 s, annealing at 61 °C for 45 s, extension at 72 °C for 2 min, and a further 10 min at 72 °C. RT-PCR products were analyzed in a 1.0% agarose gel. For PCR amplification, the primers were shown in Table 1 .
Cycler apparatus
Manufactured by Bio-Rad
Sourced in United States, China
The Cycler apparatus is a laboratory instrument designed for performing polymerase chain reaction (PCR) amplification of DNA samples. It precisely controls temperature cycles to facilitate the various steps of the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
Realmastermix kit, by Tiangen Biotech (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Plant rna reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix kit, by Takara Bio (1 mentions)
Reverse transcription system, by Promega (1 mentions)
3 protocols using cycler apparatus
1
RNA Extraction and RT-PCR Amplification
2
Quantitative Reverse Transcription-PCR Analysis
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For quantitative reverse transcription–PCR (qRT–PCR) analysis, whole seedlings or root tip tissues were harvested and frozen in liquid nitrogen for RNA extraction. RNA was extracted using the RNeasy kit (Qiagen). First-strand cDNA was synthesized from 2 μg of total RNA using Superscript III reverse transcriptase (Invitrogen) and oligo(dT) primers. qRT–PCRs were performed using a cycler apparatus (Bio-Rad) and the RealMasterMix kit (SYBR Green, Tiangen) according to the manufacturer’s instructions. The expression levels of the target genes were normalized to those of ACTIN2. Statistical significance was evaluated by Student’s t-test. The primers used to quantify gene expression levels are listed in Supplementary Table S1 at JXB online.
3
Abiotic Stress Response Profiling of Wheat
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Seeds of wheat cv. Annong 0711 were grown hydroponically in a light chamber at 25 ℃. Ten-day-old seedlings were subjected to various abiotic stresses. Seedlings were exposed to air on filter paper for induction of rapid drought conditions. Cold treatments were performed by transferring the seedlings to a pre-cooled medium in a growth chamber at 4 ℃. For drought, salt, and ABA treatments, seedlings were transferred to growth media supplemented with 20% PEG6000, 100 mM NaCl, and 6, 12, and 24 h after treatment; they were frozen immediately in liquid nitrogen and stored at -80 ℃ for RNA extraction.
For real-time PCR analysis, Total RNAs were extracted with Plant RNA Reagent (Invitrogen) and used to synthesize cDNA with a Reverse Transcription System (Promega). The expression analysis of TaCOBL was performed on a cycler apparatus (Bio-Rad) with using the SYBR Green PCR master mix kit (TaKaRa Biotechnology Co., Ltd., Dalian, China; Product Code: DRR041A). The primer sets TaCOBL-2 and Actin-p (Table S2) were used for amplification of the TaCOBL-5B and actin genes, respectively. Three replicates were performed to obtain the average and standard deviation of the expression level. Quantification of the target gene expression was carried out by the comparative CT method [34] .
For real-time PCR analysis, Total RNAs were extracted with Plant RNA Reagent (Invitrogen) and used to synthesize cDNA with a Reverse Transcription System (Promega). The expression analysis of TaCOBL was performed on a cycler apparatus (Bio-Rad) with using the SYBR Green PCR master mix kit (TaKaRa Biotechnology Co., Ltd., Dalian, China; Product Code: DRR041A). The primer sets TaCOBL-2 and Actin-p (Table S2) were used for amplification of the TaCOBL-5B and actin genes, respectively. Three replicates were performed to obtain the average and standard deviation of the expression level. Quantification of the target gene expression was carried out by the comparative CT method [34] .
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