Total RNA extraction of plant samples was performed using the Eastep®Super Total RNA Extraction Kit (Qiagen, Promega, Shanghai, China). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix with the gDNA Remover Kit (Toyobo, Osaka, Japan). Real-time RT-PCR was performed using the ABI7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) with the SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The AtUBC9 and BnaEF1-a genes were used as reference genes. The relative expression levels of the target genes were calculated by the 2−∆∆CT method [51 (link)]. All the primers used in this study are listed in Table S1 .
Sybr premix ex taqtm 2
Manufactured by Toyobo
Sourced in United States, Switzerland
SYBR Premix Ex Taq II is a real-time PCR (polymerase chain reaction) premix solution that contains SYBR Green I dye and Taq DNA polymerase. It is designed for sensitive and reliable quantification of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Abi 7300 real time detection system, by Thermo Fisher Scientific (4 mentions)
Primescript rt reagent kit, by Toyobo (3 mentions)
Eastep super total rna extraction kit, by Qiagen (2 mentions)
Gdna remover kit, by Toyobo (2 mentions)
Revertra ace qpcr rt master mix, by Toyobo (2 mentions)
Total rna kit, by Tiangen Biotech (2 mentions)
Lightcycler 96 system, by Roche (2 mentions)
Primescript rt master mix, by Toyobo (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Mirna first strand cdna synthesis tailing method kit, by Sangon (1 mentions)
Primescript rt reagent kit, by Promega (1 mentions)
Total rna kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using sybr premix ex taqtm 2
1
Plant Total RNA Extraction and RT-qPCR Analysis
2
Evaluating Expression of MtERF Genes
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Total RNA was isolated using the total RNA kit (Tiangen, Beijing, China) and then reverse transcribed into cDNA using the PrimeScript RT reagent Kit (Toyobo, Shanghai, China). qRT–PCR was performed using ABI 7300 Real-time Detection System (Applied Biosystems, USA) with SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The PCR conditions were set as follows: 95°C for 2 min; 40 cycles of 95°C for 30 s and 55°C for 30 s; and 72°C for 1 min, and the experiments were repeated three biological replicates. The ΔΔCT method was used to calculate relative expression levels of MtERF genes using GAPDH as reference gene. Primers of nine MtERF genes (randomly selected from DREB subfamily) and GAPDH gene used for qRT-PCR detection are listed in Table S1 .
3
Validating RNA-seq with qRT-PCR in Alfalfa
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To validate RNA-seq results, qRT-PCR analysis was performed as follows: first,
total RNA was isolated from alfalfa samples as previously described, and cDNA
was synthesized using the PrimeScript RT reagent Kit (Toyobo, Shanghai, China).
Reat-time qRT-PCR detection was performed on a LightCycler 96 System (Roche,
Rotkreuz, Switzerland) using SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China)
according to the manufacturer’s protocol. The PCR program was set 95 °C for 2
min, followed by 40 cycles of 95 °C for 30 s and 55 °C for 30 s, and a final
step of 72 °C for 1 min. The experiments were repeated as three biological
replicates, each run as three technical replicates. Ten MsERF genes were
randomly selected and their primers were designed for qRT-PCR detection
(Table
S1 ). Relative expression of the 10 MsERF
genes was calculated and determined based on the 2-ΔΔCT method using
GAPDH as reference gene.
total RNA was isolated from alfalfa samples as previously described, and cDNA
was synthesized using the PrimeScript RT reagent Kit (Toyobo, Shanghai, China).
Reat-time qRT-PCR detection was performed on a LightCycler 96 System (Roche,
Rotkreuz, Switzerland) using SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China)
according to the manufacturer’s protocol. The PCR program was set 95 °C for 2
min, followed by 40 cycles of 95 °C for 30 s and 55 °C for 30 s, and a final
step of 72 °C for 1 min. The experiments were repeated as three biological
replicates, each run as three technical replicates. Ten MsERF genes were
randomly selected and their primers were designed for qRT-PCR detection
(
S1
genes was calculated and determined based on the 2-ΔΔCT method using
GAPDH as reference gene.
4
Quantifying MYB Gene Expression in Medicago
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Ten R2R3-MYB genes were selected and quantitative primers were designed based on genes sequences, while the ACTIN and GAPDH genes served as reference genes (see Table S1 ). Total RNA was extracted from M. truncatula grown under the six conditions (control, ABA, drought, salt, cold, and freezing) using a total RNA kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The RNA extracts were reverse transcribed to cDNA, using a PrimeScript RT reagent Kit (Toyobo, Shanghai, China). qRT-PCR was performed using the LightCycler®96 System (Roche, Rotkreuz, Switzerland) with SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The experiments were repeated for three biological replicates and the PCR conditions were set as follows: 95 ºC for 2 min, 40 cycles of 95 ºC for 30 s, 55 ºC for 30 s, and 72 º for 1 min. The fold change value was calculated using the expression abundances, which based on the 2-ΔΔCT method.
5
Plant Total RNA Extraction and RT-qPCR Analysis
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Total RNA extraction of plant samples was performed using the Eastep®Super Total RNA Extraction Kit (Qiagen, Promega, Shanghai, China). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix with the gDNA Remover Kit (Toyobo, Osaka, Japan). Real-time RT-PCR was performed using the ABI7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) with the SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The AtUBC9 and BnaEF1-a genes were used as reference genes. The relative expression levels of the target genes were calculated by the 2−∆∆CT method [51 (link)]. All the primers used in this study are listed in Table S1 .
6
RNA extraction and RT-qPCR protocol
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TRIzol® reagent (Thermo Fisher Scientific, Inc) was used for RNA extraction when cell confluence was 70-80%. Prime Script RT Master Mix (Toyobo Life Science) was used for mRNA reverse transcription. Reverse transcription of miRNA was performed using miRNA first-strand cDNA synthesis (tailing method) kit (Sangon Biotech, China). RT-qPCR was performed using SYBR Premix EX Taq TMII (Toyobo Life Science). RNA extraction, cDNA synthesis and qPCR were performed according to the manufacturer's protocols. PCR cycle conditions: Denaturation (95°C, 30 sec); annealing (95°C, 5 sec) and extension (60°C, 30 sec) for 40 cycles. Quantification method was ΔCt=Ct (target gene)-Ct (internal reference gene), ΔΔCt=ΔCt (experiment group)-ΔCt (control group) and final results were shown as 2ΔΔCq (12 (link)). These experiments were repeated at least 3 times. The internal references for mRNA and miRNA extracted from cells were GAPDH and U6, respectively. However, miRNAs extracted from exosomes used miR16-5p as an internal reference. The primer sequences for RT-qPCR are given in Table SI .
7
Quantifying BnaCRF8 Gene Expression
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Total RNA was isolated using the total RNA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the Prime Script RT reagent Kit (Promega, Madison, WI, USA). Real-time RT-PCR was performed using ABI7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The PCR conditions were set as follows: 95 °C for 2 min; 40 cycles of 95 °C for 30 s and 60 °C for 30 s; and 72 °C for 1 min. The 2−∆∆CT method was used to calculate the relative expression levels of BnaCRF8 genes using Actin as the reference gene. The primers of the 4 BnaCRF8 genes and the Actin gene used for qRT-PCR detection are listed in Table S2 .
8
Quantitative PCR Analysis of Gene Expression
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TRIZOL Reagent (SIGMA, USA) was used for the RNA extraction in accordance with the manufacturer's instruction. Reverse transcription was conducted using Prime Script RT Master Mix (TOYOBO, Japan) in accordance with the standard protocol. SYBR Premix EX Taq TMII (TOYOBO, Japan) was used for RT-PCR. The differences in relative expression levels between groups were analyzed using the 2 -∆∆Ct method. β-actin was used as the internal control. The primer sequences used for RT-PCR were shown in Supplementary Table 1.
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