Total RNA of the collected glioma tissues and normal tissue samples was extracted by TRIzol kit (Accurate Biology, China, AG21101) and RNA purification kit (Thermo Scientific, K0731), reverse transcribed to cDNA by RT-qPCR kit (Thermo Scientific, K16225). Amplification was performed according to the SYBR green (Bio-Rad, #1725274) method with three replicate wells per sample in a total reaction system of 20 μl. PCR reaction conditions (ABI 7300 Real-Time System) were as follows: 95°C for 15 min, 40 cycles of amplification at 95°C for 15 s, 60°C for 1 min. β-Actin was used as an internal reference and 2-ΔΔCt formula was used to calculate the relative expression of the targeted gene. Targeted gene primer sequences were as follows:MLLT11: (forward) 5’-GTAGCCAGTACAGTTCCTTTCT-3’, (reverse) 5’-AAGTTGAAGGTGCTGTACTCAA-3’; ARG1: (forward) 5’-GGACCTGCCCTTTGCTGACATC-3’, (reverse) 5’-TCTTCTTGACTTCTGCCACCTTGC-3’; CD206: (forward) 5’-TCCGACCCTTCCTTGACTAATCCTC-3’, (reverse) 5’-AGTATGTCTCCGCTTCATGCCATTG-3’; IL-10: (forward) 5’-GTTGTTAAAGGAGTCCTTGCTG-3’, (reverse) 5’-TTCACAGGGAAGAAATCGATGA-3’; CD163: (forward) 5’-ATCAACCCTGCATCTTTAGACA-3’, (reverse) 5’-CTTGTTGTCACATGTGATCCAG-3’; CD115: (forward) 5’-GTCCTGAAGGTGGCTGTGAAGATG-3’, (reverse) 5’-GCTCCCAGAAGGTTGACGATGTTC-3’; PDGFβ: (forward) 5’-TCTCTGCTGCTACCTGCGTCTG-3’, (reverse) 5’-AAGGAGCGGATCGAGTGGTCAC-3’.
Rt qpcr kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The RT-qPCR kit is a laboratory instrument designed for the amplification and quantification of RNA targets through reverse transcription and real-time PCR technology. It enables researchers to detect and measure specific RNA sequences in a sample.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (15 mentions)
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (8 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Am1561, by Thermo Fisher Scientific (2 mentions)
Trizol kit, by Thermo Fisher Scientific (2 mentions)
7300 real time system, by Thermo Fisher Scientific (1 mentions)
Rna purification kit, by Thermo Fisher Scientific (1 mentions)
Trizol kit, by Accurate Biology (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Allprep dna rna mirna universal kit, by Qiagen (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqmantm microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Pcr primers, by Sangon (1 mentions)
First strand synthesis kit, by Transgene (1 mentions)
Verso 1 step rt qpcr kit, by Thermo Fisher Scientific (1 mentions)
36 protocols using rt qpcr kit
1
Quantitative Expression Analysis of Glioma Markers
2
Hippocampal RNA Extraction and qPCR Analysis
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TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from isolated hippocampal tissue, and the extracted high-quality RNA was verified using ultraviolet analysis and formaldehyde denaturation electrophoresis. PCR primers were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) [Table 1 ]. According to the instructions of a RT-qPCR kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), the fluorescent qPCR reaction was conducted with glyceraldehyde-3-phosphate dehydrogenase as an internal reference. The amplification and dissolution curves were verified after the reaction. The relative expression was calculated using the 2–ΔΔCt method.
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted from cells using TRIzol one-step reagent (Invitrogen), and then the concentration and purity of RNA were determined using UV analysis and formaldehyde deformation electrophoresis. The fluorescent qPCR reaction was performed on the instructions of the RT-qPCR kit (Thermo Fisher scientific). Primers (Table 1 ) were designed and synthesized by Sangon Biotech (Shanghai, China). Amplification curve and dissolution curve were confirmed after reaction. The relative expression of genes was calculated by 2−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference.
4
Quantitative RT-qPCR Analysis of Gene Expression
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The total RNA from tissues or cells was extracted with TRIzol reagents (16096020, Thermo Fisher Scientific Inc., Waltham, MA, United States) and reverse transcribed into complementary DNA (cDNA) using the reverse transcription kit (4368813, Thermo Fisher Scientific Inc., Waltham, MA, United States). The RT-qPCR kit (11732020, Thermo Fisher Scientific Inc., Waltham, MA, United States) was employed to perform RT-qPCR assay following the manufacturer’s protocol. The mRNA expression of each sample was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Relative mRNA expression was measured using the formula 2–ΔΔC. The primer sequences were synthesized by TaKaRa company (Takara Holdings Inc., Kyoto, Japan) (Table 1 ). Each reaction was performed in triplicate.
5
Analysis of Lymphocyte Gene Expression
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Total RNA from PBMCs was extracted using an AllPrep RNA/DNA/miRNA Universal kit (Qiagen AB, Sollentuna, Sweden) according to the manufacturer's instructions. cDNA was synthesized using an RT-qPCR kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). qPCR was performed on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The sequences of the primers used were as follows: Bcl-6, forward 5′-AGTTTATTAAGGCCAGTGA-3′ and reverse 5′-GATAGGCCATGATGTCTT-3′; c-Maf, forward 5′-ACTGGCAATGAGCAACTCCG-3′ and reverse 5′-GCTGATGATGCGGTCGGTCT-3′; B lymphocyte-induced maturation protein-1 (Blimp-1), forward 5′-TCCAGCACTGTGAGGTTTCA-3′ and reverse 5′-TCAAACTCAGCCTCTGTCCA-3′; and PD-1, forward 5′-GACAACGCCACCTTCACCT-3′ and reverse 5′-GCTTGTCCGTCTGGTTGCT-3′. GAPDH, forward 5′-AATCCCATCACCATCTTCCA-3′ and reverse 5′-TGGACTCCACGACGTACTCA-3′. qPCR was performed with SYBR Select Master Mix (Thermo Fisher Scientific Inc.) and 500 nM forward and reverse primers. Thermal cycle conditions used were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 1 sec and 60°C for 30 sec Gene expression values were calculated by the comparative threshold cycle method (11 (link)). Stable expressed GAPDH served as endogenous control.
6
Robust miRNA and mRNA Quantification
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Total RNA was extracted using a Trizol kit, and then reversely transcribed into complementary DNA (cDNA) using TaqManTM MicroRNA Reverse Transcription Kit (for miRNA, 4366596, Thermo Fisher Scientific Inc., Waltham, MA, United States) and High-Capacity cDNA Reverse Transcription Kit (for mRNA, 4368813, Thermo Fisher Scientific Inc., Waltham, MA, United States). The RT-qPCR experiment was carried out according to the instructions of the RT-qPCR kit (11732020, Thermo Fisher Scientific Inc., Waltham, MA, United States). The primer sequences were designed and provided by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China) (Table 1 ). With U6 as the internal reference for miR-511-3p and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal reference for Six3os1 and Fezf1, the fold changes of target genes between experimental group and control group were calculated by the 2–ΔΔCt relative quantification method.
7
Quantifying mRNA and miRNA Transcripts
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Total RNA content of tissues and cells was extracted with TRIzol (16096020; Thermo Fisher Scientific). RNA of miRNA was reversely transcribed using reverse transcription kit TaqMan ™ MicroRNA Reverse Transcription Kit (4366596; Thermo Fisher Scientific) and the RNA of mRNA was reversely transcribed using reverse transcription kit High‐Capacity cDNA Reverse Transcription Kit (4368813; Thermo Fisher Scientific). The primer sequences of lincRNA‐p21, miR‐221, SIRT1, Pcsk9, GAPDH and U6 were designed and provided by Sangon Biotech. The primer sequences are shown in Table S1 , with GAPDH as internal reference. Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) kit (11732020; Thermo Fisher Scientific) was used to carry out the RT‐qPCR experiment according to the instructions using 2 ‐ Δ Δ Ct
8
RNA Extraction and RT-qPCR Analysis
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The one-step method of TRIzol (Invitrogen, Carlsbad, CA, USA) was employed to extract total RNA, and extracted high-quality RNA was confirmed using ultraviolet analysis and formaldehyde denaturation electrophoresis. RT-qPCR was conducted as per the instructions of a RT-qPCR kit (Thermo Fisher Scientific, Shanghai, China) with glyceraldehyde-3-phosphate dehydrogenase as an internal reference for CCL22, peroxisome proliferator-activated receptor gamma (PPARγ), PVT1, and SOCS5, while U6 as an internal reference for miR-21-5p. PCR primers were provided by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table 1 ). The amplification and dissolution curves were confirmed after the reaction, and data were analyzed by 2−ΔΔCt method.
9
Quantitative Analysis of miRNA and mRNA
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Total tissue or cellular RNA was extracted using Trizol (16,096,020, Thermo Fisher Scientific, Rockford, IL). miRNA and mRNA were reversely transcribed using the TaqMan™ MicroRNA Reverse Transcription Kit (4,366,596, Thermo Fisher Scientific) and the High-Capacity cDNA Reverse Transcription Kit (4,368,813, Thermo Fisher Scientific), respectively. The primer sequences were designed and provided by Sangon Biotech (Shanghai, China), as shown in Table S1 . U6 was used as the internal reference for miR-22-3p and GAPDH for c-fos, MALAT1 and STAT1. RT-qPCR was completed with the RT-qPCR kit (117,320, Thermo Fisher Scientific). The 2−ΔΔCt was adopted for gene expression analysis.
10
Quantitative RNA Expression Analysis
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TRIzol (Invitrogen) one-step method was applied to extract total RNA of cells and tissues. Next, the extracted qualified RNA was tested by ultraviolet analysis and formaldehyde denaturation electrophoresis. The fluorescent quantitative PCR was conduct based on instructions of RT-qPCR kit (ThermoFisher). PCR primers were made and synthesized in Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table 2 ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized as an internal references of YAP1, and U6 as an internal reference of miR-375. After reaction, amplification curves and dissolution curve were identified. The relative expression was calculated by 2−ΔΔCt method.
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