Total RNA extracted from cells with the Qiagen miRNeasy Mini kit (Qiagen) was reversely transcribed to complementary DNA by using the TaqMan miRNA RT Kit and stem-loop RT primers (Applied Biosystems). MiRNA expression levels were tested using the TaqMan PCR kit as implemented in the ABI 7900 real-time PCR System (Applied Biosystems). To assess the mRNA expression levels of miR-155-5p target genes, SYBR PCR Master Mix reagent kits (TaKaRa) were used according to the manufacturer's instructions. The results of miRNA and mRNA expression were normalized using the threshold cycle (Ct) of U6 and β-actin, respectively. All reactions, including no-template controls, were performed in triplicate. Specific primers for amplification are shown in Supplementary Table S7 .
Stem loop rt primer
Manufactured by Thermo Fisher Scientific
Sourced in China, United States, Japan
The Stem-loop RT primer is a laboratory tool used for the detection and quantification of specific microRNA (miRNA) molecules. It is designed to reverse-transcribe miRNA into complementary DNA (cDNA) for downstream analysis, such as real-time PCR. The primer contains a stem-loop structure that binds to the 3' end of the miRNA, allowing for specific and efficient reverse transcription.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Taqman mirna probes, by Thermo Fisher Scientific (10 mentions)
Amv reverse transcriptase, by Takara Bio (10 mentions)
Taqman pcr kit, by Thermo Fisher Scientific (9 mentions)
Applied biosystems 7300 sequence detection system, by Thermo Fisher Scientific (5 mentions)
Taqman probe, by Thermo Fisher Scientific (5 mentions)
Reverse transcriptase, by Takara Bio (4 mentions)
Taqman mirna rt kit, by Thermo Fisher Scientific (3 mentions)
Abi 7900 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Mirneasy mini kit, by Qiagen (3 mentions)
Avian myeloblastosis virus reverse transcriptase, by Takara Bio (3 mentions)
Taqman mirna pcr kit, by Thermo Fisher Scientific (3 mentions)
Taqman mirna assay, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 7900 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dntps, by Takara Bio (2 mentions)
Rnase inhibitor protein, by Takara Bio (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Sybr pcr master mix reagent kit, by Takara Bio (1 mentions)
Taqman universal master mix, by Roche (1 mentions)
35 protocols using stem loop rt primer
1
Quantitative Analysis of miRNA Expression
2
Quantification of sncRNA715 in Mice Sciatic Nerve
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Sciatic nerves were prepared from P 1, 4 or 9 C57BL/6J mice as described above, dissociated in 700μl Qiazol using a Tissue Ruptor (Qiagen) and total RNA was extracted using the miRNeasy Mini Kit (Qiagen). Reverse transcription of mRNAs was performed with the Transcriptor High Fidelity Reverse Transcription Kit and qPCR was performed with the Taqman Universal Master Mix (all Roche Applied Science). SncRNA715 was reverse transcribed by the TaqMan MicroRNA Reverse Transcription Kit with stem-loop RT primers specific for sncRNA715 or snoRNA135 sequence (Applied Biosystems, order no. sncRNA715 PN4427975, snoRNA135 PN440887) and amplified with the Taqman Universal Master Mix (Roche Applied Sience) with specific primers and probes for the indicated sncRNAs (Applied Biosystems). The crossing points were used for relative quantification based on the ΔΔCt method using REST software [27 ]. SnoRNA135 was used as a reference gene. PCR products and the 10 bp DNA Step Ladder (Promega) were separated on 4% agarose gels and stained with ethidium bromide.
3
Extraction and Profiling of miRNAs from Cell-Derived Microvesicles
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The total RNA of the MVs derived from 108 cells was extracted using TRIzol Reagent (Life Technologies). Samples of total RNA were extracted for miRNAs Affymetrix GeneChip miRNA 3.0 array analysis (Life Technologies). Quantitative real-time PCR (qPCR) was performed using TaqMan microRNA probes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, 1 μg of the total RNA was reverse-transcribed to produce cDNA using Avian Myeloblastosis Virus reverse transcriptase (Takara, Dalian, China) and stem-loop RT primers (Applied Biosystems). Real-time PCR was performed using a TaqMan PCR kit and an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). All of the reactions, including the no-template controls, were run in triplicate. After the reactions, the CT values were determined using fixed-threshold settings.
4
Quantification of miRNA Expression in RCC CD8+ T Cells
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CDNA was synthesized from total RNA of RCC CD8+ T cells using gene-specific primers according to theTaqMan MicroRNA assay protocol (PE Applied Biosystems, Foster City, CA). Reverse reactions for miR-29b and miR-198 were performed using 10 ng of RNA samples, 50 nM stem-loop RT primers, 1× RT buffer, 0.25 mM each of dNTPs, 3.33 U/μl multiscribe reverse transcriptase and 0.25 U/μl RNAse inhibitor (all purchased from cDNA Archive kit of Applied Biosystems). Real-time PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR system. The 10 μl PCR volume included 0.67 μl of RT product, 1× TaqMan universal PCR master mix and 1 μl of primers and probe mix of the Taq-Man MicroRNA assay (PE Applied Biosystems). Ct were determined using default threshold settings. miRNA let-7a was identified as the most stable housekeeping miRNA in lymphmonocytes and used to normalize the expression of each miRNA [24 (link)]. Relative quantification (RQ) of miRNA expression was calculated with the 2-ΔΔCt method (Applied Biosystems User Bulletin N°2 [P/N 4303859]). Data are presented as relative quantity (RQ) of target miRNA, normalized with respect to miR-let-7a and a calibrator sample. As a calibrator, we used CD8+ T cells isolated from healthy normal donors.
5
Quantification of miRNA Expression
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Total RNA from four types of cells was extracted by using the miRNeasy Mini kit (Qiagen, Germany) and then was reversely transcribed to complementary DNA with the TaqMan miRNA RT Kit and stem‐loop RT primers (Applied Biosystems, USA). miRNA expression was detected based on the TaqMan PCR kit implemented in the ABI 7900 real‐time PCR System (Applied Biosystems, USA) and normalized using the threshold cycle (Ct) of U6. All reactions were performed in triplicate.
6
Quantification of miR-33a in NSCLC
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Total RNA was extracted from NSCLC and normal tissues using TRIZOL reagent, according to manufacturer’s instructions. RNA concentration was measured by a spectrophotometer, and the quality of all RNA samples was assessed by electrophoresis on 1.5% denaturing agarose gels. qRT-PCR was carried out using a Taqman miRNA PCR kit (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. Briefly, total RNA was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China) and the stem- loop RT primers (Applied Biosystems). Real-time PCR was performed using TaqMan miRNA probes on the Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). U6 was used as the internal control. The 2-δδCT method was used to quantify the expression levels of miR-33a, and the expression status (e.g., high levels or low expression levels) was recorded.
7
Plasma miRNA profiling for prognosis
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From 400-µl samples of plasma, total RNA was purified using a miRNeasy Serum/Plasma kit (Qiagen, Tokyo, Japan) according to the manufacturers protocol. miR-191 was used as an endogenous internal control (28 (link),29 (link)).
We used TaqMan miRNA probes (Applied Biosystems Japan Ltd., Tokyo, Japan) to perform the qRT-PCR assay according to the manufacturers instructions. In each step, from plasma purification to the qRT-PCR, an equal volume (400 µl) of plasma sample was processed. The total RNA was reverse-transcribed to complementary DNA using the TaqMan miRNA Reverse Transcription kit (Applied Biosystems) and stem-loop RT primers (hsa-miR-135, hsa-miR-6826, hsa-miR-6835 and hsa-miR-6875, in addition to hsa-miR-191 for the internal control) (Applied Biosystems). RT-PCR was performed using the LightCycler® 480 System II (Roche Diagnostics K.K., Tokyo, Japan). The reactions were initiated at 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. All reactions, including the no template controls, were run in duplicate. The relative expression levels of the target miRNAs were normalized to those of miR-191 according to the ΔΔCt method. For every target miRNA, the relative Ct values were divided into an OS ≥2-year group (responders) and an OS <2-year group (non-responders) which were plotted separately.
We used TaqMan miRNA probes (Applied Biosystems Japan Ltd., Tokyo, Japan) to perform the qRT-PCR assay according to the manufacturers instructions. In each step, from plasma purification to the qRT-PCR, an equal volume (400 µl) of plasma sample was processed. The total RNA was reverse-transcribed to complementary DNA using the TaqMan miRNA Reverse Transcription kit (Applied Biosystems) and stem-loop RT primers (hsa-miR-135, hsa-miR-6826, hsa-miR-6835 and hsa-miR-6875, in addition to hsa-miR-191 for the internal control) (Applied Biosystems). RT-PCR was performed using the LightCycler® 480 System II (Roche Diagnostics K.K., Tokyo, Japan). The reactions were initiated at 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. All reactions, including the no template controls, were run in duplicate. The relative expression levels of the target miRNAs were normalized to those of miR-191 according to the ΔΔCt method. For every target miRNA, the relative Ct values were divided into an OS ≥2-year group (responders) and an OS <2-year group (non-responders) which were plotted separately.
8
Quantifying Serum and Tissue miRNA
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qPCR for individual miRNAs was performed on independent sets of serum or tissue using a two-step procedure. qPCR for miRNA Stem-Loop™ RT primers for miR-483-5p and miR-16-5p were synthesized by Applied Biosystems (Thermo Fisher Scientific, Inc.) (Table I ). A PrimeScript™ RT reagent kit (Perfect Real Time; Takara Biotechnology Co., Ltd., Dalian, China) was used to reverse transcribe the total RNA. A SYBR Green (Takara Biotechnology Co., Ltd.) qPCR assay kit was used to detect the expression of miR-483-5p and miR-16-5p. The qPCR reaction was performed over 45 cycles (95°C, 10 sec; 60°C, 30 sec) following an initial denaturation step (95°C, 5 min) on the CFX96 system using Bio-Rad CFX Manager 2.0 Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The expression levels of miRNA were calculated and quantified using the 2−ΔΔCq method (17 (link)). miR-16-5p was used as the internal control. All reactions were performed in triplicate.
9
Plasma miRNA Quantification Using TaqMan Assay
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TaqMan miRNA Assay (Applied Biosystems) was used to quantify mature miRNAs in plasma samples in accordance with the manufacturer's instructions. Briefly, 2 mL of total RNA was reverse-transcribed to cDNA using M-MLV reverse transcriptase (Promega, Madison, Wisc.) and stem-loop RT primers (Applied Biosystems). Real-time PCR was performed using TaqMan miRNA probes (Applied Biosystems) on the CFX-96 system (Bio-Rad, Hercules, Calif.). All reactions, inthe no-template controls, were run in triplicate. After real-time PCR amplification, the Ct values were determined using the fixed threshold settings. Each miRNA was reverse transcribed and amplified separately.
10
Quantitative miRNA Expression Analysis
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For qRT-PCR, the total RNA was reverse transcribed to complementary DNA by using the TaqMan miRNA RT Kit and stem-loop RT primers (Applied Biosystems, USA). The quantitative detection of miRNA was performed using the TaqMan PCR kit as implemented in the ABI 7900 Real-Time PCR System (Applied Biosystems, USA). The reactions were initiated with a 384-well optical plate at 95° for 5 min, followed by 40 cycles of 95° for 15 s and 60° for 1 min [30] . Equal amount of tumor tissue and adjacent non-tumor tissue was assigned in each plate and the expression levels of target miRNAs and internal control miRNA (RNU43) were measured simultaneously. All reactions were performed in triplicate. The relative expression levels of target miRNAs were calculated with 2 -ΔΔCT , where CT means cycle threshold and its values were obtained from Bio-Rad iQ5 2.1 Standard Edition Optical System Software.
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