Total RNA was obtained. Primers (Table 1 ) were designed and synthesized by Takara (Takara. Kyoto, Japan). Reverse transcription was conducted as TaqMan MicroRNA Assays Reverse Transcription Primer (4366596, Thermo scientific, Waltham, MA, USA). Quantitative PCR was performed using SYBR® Premix Ex TaqTM II Kit (RR820A, Xingzhi Biotechnology Co., Ltd., Guangzhou, Guangdong, China) in the ABI PRISM® 7300 system (Prism® 7300, Shanghai Kunke Instrument Equipment Co., Ltd., Shanghai, China). U6 served as an internal control for miR‐206 and GAPDH (abs830032, Absin Bioscience Inc, Shanghai, China) for others. The 2−ΔΔCt formula was used.
Taqman microrna assays reverse transcription primer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan MicroRNA Assays Reverse Transcription primer is a component used in the quantification of microRNA (miRNA) expression levels. It is designed to facilitate the reverse transcription of miRNA molecules into cDNA, which can then be amplified and detected using real-time PCR techniques.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Primescript rt reagent kit, by Takara Bio (8 mentions)
Nanodrop 2000 micro ultraviolet spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Trizol kit, by Thermo Fisher Scientific (4 mentions)
Abi7500 quantitative pcr instrument, by Thermo Fisher Scientific (3 mentions)
Taqman multiplex real time solution, by Thermo Fisher Scientific (2 mentions)
Primescript rt kit, by Takara Bio (2 mentions)
Abi 7500 instrument, by Thermo Fisher Scientific (2 mentions)
Fast sybr green pcr kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Abs830032, by Absin (1 mentions)
Rna extraction kit, by GenStar (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Abi 7500 qpcr instrument, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Takara Bio (1 mentions)
34 protocols using taqman microrna assays reverse transcription primer
1
Quantification of miRNA Expression
2
Endometrial Gene Expression Analysis
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The endometrial tissues and cells from the BALB/c mice of each group were extracted, and the total RNA was extracted in strict accordance with the provided instructions of an ultrapure RNA extraction kit (Qiagen, Hilden, Germany). At the tissue level, the expression of EPHA3 and mTOR were determined. The primers of EPHA3, mTOR, autophagy-related gene 3 (Atg3), light chain 3 (LC3), Beclin1, bax, fas and B-cell lymphoma-2 (bcl-2) were synthesized by Aoke Biotechnology Co., Ltd. (Zhenjiang, Jiangsu, China) (Table 1 ). Next, the RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFi-MMLV and RNase-free water were all dissolved on ice for later use. The extracted RNA (20 μl) was reverse transcribed in accordance with the instructions of TaqMan MicroRNA Assays Reverse Transcription Primer (4366596, Thermo Scientific, Waltham, MA, U.S.A.). The quantitative PCR was conducted in strict accordance with the instructions of the SYBR® Premix Ex Taq™ II Kit (RR820A, Xingzhi Biotechnology Co., Ltd., Guangzhou, Guangdong, China) on an ABI PRISM® 7300 (Prism® 7300, Shanghai Kunke Instrument Equipment Co., Ltd., Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, abs830032, ABX Biotechnology Co., Ltd., Shanghai, China) was regarded as an internal reference, and the fold changes were calculated by means of relative quantitation (2−ΔΔCt method).
3
Quantifying miRNA and mRNA Expression
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After the total RNA was extracted using Trizol (Thermo Scientific, USA), the TaqMan MicroRNA Assays Reverse Transcription Primer (Thermo Scientific, USA) was used to synthesize cDNA. A SYBR® Premix ExTaqTMII kit (Xingzhi Biotech Co., Ltd., China) was adopted to perform a qPCR on an ABIPRISM®7300 system (Prism®7300, Shanghai Kunke Instrument Equipment Co., Ltd., Shanghai, China). The internal control of miR-23a was U6, and that of TGFBR2 was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with 2–ΔΔCt used to represent the relative expression of genes to be tested. The primers are shown in Table 1 .
4
Quantitative Analysis of miR-124-3p Expression
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After 48 h of transfection, cells were harvested from each group. Total RNAs were extracted using Trizol (16096020 from Thermo Fisher Scientific; and B1802 from HaiGene Bio, Quezon City, Philippines) followed by reverse transcription into cDNAs using TaqMan MicroRNA Assays Reverse Transcription Primer (Thermo Fisher Scientific). Next, SYBR ® Premix ExTaq TM II kit (Xingzhi Biotech, Guangzhou, China) was used for quantitative real-time polymerase chain reaction (qRT-PCR). Reagents were added in the following order: SYBR ® Premix ExTaq TM II (2×, 25 μL), forward primer (2 μL), reverse primer (2 μL), ROX Reference Dye (50×, 1 μL), DNA template (4 μL), and double distilled H 2 O (16 μL). An ABI Prism ® 7300 fluorescence quantitative PCR instrument (Shanghai Kunke Instrument & Equipment Co., Shanghai, China) was used to conduct qRT-PCR with the following running parameters: 95°C for 10 min (pre-denaturation), 32 cycles of 95°C for 15 s (denaturation) and 60°C for 30 s (annealing) before extension at 72°C for 1 min. ΔCt = Ct target gene -Ct GAPDH; ΔΔCt = ΔCt study group -ΔCt control group . U6 and GAPDH were the internal controls for miR-124-3p and other genes, respectively. The relative gene expression of the target gene was calculated using the 2 -ΔΔCt method. The primers are listed in Table 1.
5
Quantitative RT-PCR of miRNA Transcripts
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Total RNA was extracted with RNA Extraction Kit (D203-01, GenStar Biosolutions Co., Ltd., Beijing, China) and then reversely transcribed as per the instructions of TaqMan MicroRNA Assays Reverse Transcription Primer (4366596, Thermo Fisher Scientific Inc., Waltham, MA). RT-qPCR was conducted using SYBR® Premix Ex TaqTM II kit (RR820A, Action-award Biological Technology Co., Ltd, Guangzhou, China) on the ABI PRISM® 7300 system (Prism® 7300, Shanghai Kunke Instrument Equipment Co., Ltd., Shanghai, China). Takara Biotechnology Ltd. (Dalian, China) designed and synthesized the primers, with sequences shown in Supplementary Table 3 . The fold changes were calculated using relative quantification (the 2−ΔΔCt method), with GAPDH as a loading control.
6
Quantitative PCR Analysis of RNA Expression
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Total RNA was extracted from brain tissues using TRIzol (Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using TaqMan MicroRNA Assays Reverse Transcription Primer (Thermo Fisher Scientific, Inc.). Quantitative PCR was performed using SYBR® Premix Ex Taq™ II Kit (Takara). The following components were added in the mixture: 25 µl of SYBR® Premix Ex Taq™ II (2x), 2 µl of PCR upstream and downstream primers, 1 µl of ROX Reference Dye (50x), 4 µl of DNA template and 16 µl of ddH2O. Fluorescence quantitative PCR was performed with ABI PRISM® 7300 (Kunke Equipment Co., Ltd., Shanghai, China). The reaction conditions were as follows: 10 min pre-denaturation at 95˚C, followed by 32 cycles at 95˚C for 15 sec and 60˚C for 30 sec, and 72˚C for 1 min. The relative expression levels were normalized to endogenous control U6 and were expressed as 2-ΔΔCq and calculated as follows (28 (link)): ΔCt=Ct (target gene)-Ct (U6) and ΔΔCt=ΔCt (experimental group)-ΔCt (control group). The sequences of the primers used are present in Table I .
7
Corneal Gene Expression Analysis
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Six mice in each group were randomly euthanized 14 days after the operation, and corneas were collected and put into a 1.5 mL centrifuge tube. The total RNA was extracted using Trizol kits (Invitrogen) and then reverse-transcribed into complementary DNA (cDNA) according to the instructions of TaqMan microRNA Assays Reverse Transcription Primer (4427975, Applied Biosystems, Carlsbad, CA, USA). Next, 5 μL of cDNA products were obtained as a template for PCR amplification. β-Actin was used as an internal parameter for mRNAs. The relative difference of gene expression was calculated using 2−ΔΔCT method. The primer sequence is shown in Table S2 (primer design was carried out using the primer design function provided by NCBI).
8
miRNA Expression Analysis by RT-qPCR
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The total RNA was extracted using the miRNeasy Mini Kit (217004, Qiagen, Hilden, Germany) and reversely transcribed into complementary DNA (cDNA) with TaqMan MicroRNA Assays Reverse Transcription Primer (4427975, Applied Biosystems, Carlsbad, CA, USA). The primers were designed and synthesized by Takara (Kyoto, Japan) (Table 1 ). RT-qPCR was performed on an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA). The expression of miR-34a-5p was examined using a TaqMan microRNA assay kit (Applied Biosystems, Carlsbad, CA, USA) (Li et al., 2017 (link)). U6 was taken as the internal reference of miR-34a-5p and β-actin was the reference for other genes. The fold changes in expression were calculated by means of relative quantification 2−ΔΔCt.
9
Quantitative Analysis of EZH2, BLACAT1, CDKN1C, and CCNE
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TRIzol (Invitrogen, Calsbad, CA, United States) was used to extract total RNA from tissues and cells. Nanodrop2000 micro-ultraviolet spectrophotometer (1011U, nanodrop, United States) was used to detect the concentration and purity of total RNA. Reverse transcription was conducted to generate cDNA according to the instruction of TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, United States). The primers of EZH2, BLACAT1, CDKN1C, and CCNE were designed and then synthesized by Takara company (Table 1 ). ABI 7500 quantitative PCR instrument (7500, ABI, United States) was used for real-time fluorescence quantitative PCR detection. The reaction conditions were pre-denaturation at 95°C for 10 min, denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 34 s, with a total of 40 cycles. The relative transcription level of target gene was calculated by relative quantitative method (2-ΔΔCT method) with GAPDH as internal reference: ΔΔCt = ΔCt experimental group-ΔCt control group, ΔCt = Ct (target gene) – Ct (internal reference), relative transcription level of target gene mRNA = 2-ΔΔCt. Each experiment was repeated three times.
10
Comprehensive RNA Extraction and qPCR Analysis
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The TRIzol (Invitrogen, Carlsbad, CA, USA) method was used to extract total RNA from bone marrow blood, tissues, and cells. The NanoDrop 2000 micro ultraviolet spectrophotometer (1011U, NanoDrop Technologies, Inc., Rockland, ME, USA) was used to detect the concentration and purity of the extracted total RNA. cDNA was generated from RNA according to the manuals of TaqMan MicroRNA Assays Reverse Transcription primer (4427975, Applied Biosystems, Carlsbad, CA, USA)/PrimeScript RT reagent Kit (RR047A, Takara, Tokyo, Japan). miR-27, NEDD4, and Notch1 primers were synthesized by Takara (Table 1 ). RT-qPCR was conducted with TaqMan Multiplex Real-Time Solution (4461882, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) on ABI 7500 quantitative PCR instrument (7500, Applied Biosystems). The relative transcription level of the target gene was calculated using the relative quantitative method (2−ΔΔCT method) (18 (link)) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, for NEDD4 and Notch1) and U6 (for miR-27).
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