Total RNA was isolated from the cells using Trizol reagent (Invitrogen, USA). Purity and concentration of total RNA were measured by a Nanodrop 1000 spectrophotometer (Thermo Scientific, Rockford, IL). Reverse transcription was performed by FastKing gDNA Dispelling RT SuperMix with RNase Inhibitor (TIANGEN, Beijing). Specific forward and reverse primers (see Supplementary Materials) for each gene were constructed by BioTNT Company. Then, mRNA expression was detected using SuperReal PreMix Plus Kit (Tiangen, Beijing). Reaction was performed in a 96-well plate format using an ABI™7500 real-time PCR instrument (Applied Biosystems). Ct values were converted to comparative Ct values (2−ΔΔCt) by comparison to reference gene GAPDH.
Superreal premix plus kit
Manufactured by Tiangen Biotech
Sourced in China, United States
The SuperReal PreMix Plus kit is a laboratory equipment product designed for real-time quantitative PCR (qPCR) applications. It contains a pre-mixed solution that includes all the necessary components for efficient and accurate qPCR reactions, including a DNA polymerase, nucleotides, and reaction buffer.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (23 mentions)
Fastquant rt kit, by Tiangen Biotech (19 mentions)
Fastking rt kit, by Tiangen Biotech (7 mentions)
Trizol reagent, by Tiangen Biotech (7 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (6 mentions)
Cfx96 real time pcr system, by Bio-Rad (5 mentions)
Rnaprep pure tissue kit, by Tiangen Biotech (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Rna extraction kit, by Tiangen Biotech (3 mentions)
Fastquant rt kit with gdnase, by Tiangen Biotech (3 mentions)
Lightcycler 480, by Roche (3 mentions)
Minibest plant rna extraction kit, by Takara Bio (3 mentions)
Primescript rt master mix kit, by Takara Bio (3 mentions)
Mircute plus mirna first strand cdna kit, by Tiangen Biotech (3 mentions)
Mircute plus mirna qpcr kit, by Tiangen Biotech (3 mentions)
Fastking rt kit with gdnase, by Tiangen Biotech (3 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
74 protocols using superreal premix plus kit
1
Quantitative Real-Time PCR Analysis
2
RNA Extraction, cDNA Synthesis, and qPCR Workflow
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This work utilized RNA extraction kit (Tiangen DP419, Beijing, China) for isolating total RNA. Later, agarose gel electrophoresis (AGE) was conducted to detect RNA integrity, while the Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, USA) was employed to detect RNA content. Besides, the FastKing RT kit that contained gDNase (Tiangen KR116, Beijing, China) was adopted for cDNA synthesis. SuperReal PreMix Plus kit (SYBRGreen FP205, Tiangen, Beijing, China) was applied in qPCR on Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA). Conditions of the PCR procedure were shown below, 30-s under 95 °C; 5-s under 95 °C and 30-s under 60 °C for 40 cycles; and melting curve detection under 65–95 °C. The assay was carried out in triplicate independently. 2−ΔΔCt approach was adopted for determining the relative expression levels of genes, with StEF-1α (AB061263) being an internal control. Table S9 displays the primers.
3
Quantitative Analysis of miRNA Expression
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RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) and synthesized into cDNA using the FastQuant RT Kit (with gDNase) (Tiangen, Beijing, China). Quantitative real-time RT-PCR (qRT-PCR) was conducted using a SuperReal PreMix Plus Kit (SYBR Green) (Tiangen, Beijing, China). The qRT-PCR was performed on a LightCycler 480 (Roche Molecular Systems, CA, USA). Cucumber Actin was used as the reference gene to normalize the data. To validate the presence and relative expression of the miRNAs obtained from the sequencing, the known and novel miRNAs were assayed by qRT-PCR. The reverse transcription reaction was performed using a miRcute miRNA First-Strand cDNA Synthesis Kit (Tiangen, Beijing, China). The qRT-PCR was performed using the miRcute miRNA qRT-PCR Detection Kit (SYBR Green) (Tiangen, Beijing, China), and U6 snRNA was used as the internal control. The relative expression was calculated according to the 2−ΔΔCt method73 (link), and the standard deviation was calculated using three biological replicates. The primers used in the experiment were synthesized by GENEWIZ (Suzhou, China) and are listed in Supplementary Table S5 .
4
RNA Extraction and RT-qPCR Analysis
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TRIzol reagent (Invitrogen, Carlsbad, USA) was applied under RNAase free condition to extract the total RNAs from the HUVECs after 24-h treatment with the HA according to manufacturer’s instruction. The RNAs were reverse transcribed into cDNAs by a FastQuant RTkit (Tiangen Biotech Co., Ltd., Beijing, China) followed by Quantitative real-time PCR (qPCR) with the responsible primers using SuperReal PreMix Plus kit (Tiangen Biotech Co., Ltd., Beijing, China) on Roche Cobas z 480 Real-Time PCR Detection System (Roche, Basel, Switzerland). All the primers used for RT-qPCR were obtained from GeneCopoeia (GeneCopoeia Inc., Germantown, Maryland, USA). The expression of the mRNAs was normalized by expression of GAPDH mRNA as a control and calculated based on the 2−ΔΔCt method.
5
RNA Isolation and qRT-PCR Analysis
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The methods of RNA isolation and qRT-PCR were done according to Dan et al. (2021) (link) with some modifications. The plant samples in each replication were collected and homogenized in liquid nitrogen to powder. Total RNA was extracted from the homogenized powder by applying a MiniBEST PLANT RNA extraction Kit (TaKaRa). The concentration of the isolated RNA was measured by a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Then 4 µg RNA was used for producing the reverse-transcribed complementary DNA (cDNA) with Dnase I, oligo (dT) primers, dNTPs and M-MLV (TIANGEN) in a 20 µL reaction. A total of 2 µL cDNA from the above reaction was employed as a template to determine the transcript levels of the tested genes with a SuperReal PreMix Plus kit (TIANGEN) on a Roche LightCycler instrument. There were three biological replicates per treatment. The primers used for qRT-PCR were designed using primer premier 5 software, and were listed in File S1 . The tomato ACTIN gene was used to normalize relative expression levels.
6
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was extracted and reverse transcription was performed using Primescript™ RT Master Mix kit (Takara, Shiga, Japan). qRT-PCR was performed in a Chromo4 instrument cycler (Bio-Rad, Hercules, USA) using Superreal Premix plus kit (Tiangen Biotech, Beijing, China). PCR amplification was carried out with the following cycling parameters: denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s. Primer sequences (Synthesized by Sangon Biotech Co., Ltd., Shanghai, China) were listed in Tables S1 , 2.
7
Transcriptome Analysis of RNA Samples
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Total RNA from different treatments was extracted using TRIzol reagent (Life Technologies) and first-strand cDNAs were synthesized using the ReverTra Ace quantitative real-time PCR (qRT-PCR) Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. qRT-PCR was performed using the SuperReal PreMix Plus Kit (Tiangen, Beijing, China) in CFX Connect real-time PCR detection system (Bio-Rad, Hercules, CA, USA) according to the manufacturers’ instructions. Actin was used as the internal control (Primers used are listed in Supplementary Table S13
8
Quantification of miRNA and mRNA
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Total RNA was separated with Trizol reagent and reverse‐transcribed into cDNA using the miRcute Plus miRNA First‐Strand cDNA kit (Tiangen) for miRNA or FastKing RT kit (Tiangen) for mRNA. miRNA was quantified using the miRcute Plus miRNA qPCR kit (Tiangen), and mRNA was quantified using the SuperReal PreMix Plus kit (Tiangen). Gene expression was normalized to U6 or GAPDH and calculated by the 2−ΔΔCt method. See Table S1 for polymerase chain reaction (PCR) sequences.
9
Liver RNA Isolation and RT-qPCR Analysis
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Total RNA was isolated from the rat livers using TRIzol® reagent (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. The mRNA concentrations were measured using a Nano Drop spectrophotometer (Thermo Fisher Scientific, Inc.). The appropriate quantity of cDNA templates (~0.5 mg) was generated with a Fast Quant RT kit (Tiangen Biotech Co., Ltd.) at 42°C for 15 min and 95°C for 3 min, according to the manufacturer's protocol. Reverse transcription-quantitative (RT-q)PCR was performed using a SuperReal PreMix Plus kit (Tiangen Biotech Co., Ltd.) at 95°C for 15 min; followed by 40 cycles of 95°C for 10 sec and 60°C for 32 sec, according to the manufacturer's protocol (14 (link)). All samples from 6 rats per group were measured in triplicate, and the mean value was used for the comparative analysis. Quantitative measurements were calculated using the 2−∆∆Cq method (15 (link)). β-actin served as the housekeeping gene for the comparison of the gene expression data. The primer sequences used for RT-qPCR analyses were synthesized by Takara Biotechnology Co., Ltd. and are listed in Table I .
10
Ovary RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from the ovary of 8-day-old adult females using Trizol reagent (Invitrogen). First-strand cDNA was reverse transcribed with FastQuant RT Kit (with gDNase) (Tiangen). qRT-PCR was performed using a Light cycler 96 (Roche) and a SuperReal Premix Plus kit (Tiangen), initiated at 95°C for 15 min, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 30 s. Melting curve analysis was performed to confirm the specificity of amplification. Ribosomal protein 49 (rp49) was used as a reference control. The 2-ΔΔCt method was applied to calculate relative expression levels. Primers used for qRT-PCR are listed in S1 Table .
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