RNA was extracted from plasma as described in the Supplementary Materials and Methods . Five microlitres of total RNA were used for first-strand cDNA synthesis in a 15-μl reaction volume, using the TaqMan miRNA Reverse Transcription kit and miRNA-specific stem-loop primers (Thermo Fisher Scientific, Foster City, CA, USA). cDNAs (2.5 μl) were amplified for 45 cycles using TaqMan miRNA primers and probes (Thermo Fisher Scientific) and LightCycler 480 PCR Master Mix (Roche Diagnostics GmbH, Mannheim, Germany). No-RT and no-template negative controls were included. The amplification reactions were performed in a LightCycler 480 II (Roche). Signals were quantified using the second derivative maximum method (Software Version 1.5, Roche). All amplification curves were carefully examined, and products with poor curves were discarded. Ct values <40 were obtained for >99% of the reaction samples.
Mirna specific stem loop primer
Manufactured by Thermo Fisher Scientific
Sourced in United States
MiRNA-specific stem-loop primers are designed for the detection and quantification of microRNA (miRNA) expression. They are used in reverse transcription and qPCR workflows to specifically amplify mature miRNA sequences. The stem-loop structure of the primers enhances their specificity for mature miRNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (23 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Mirvana paris kit, by Thermo Fisher Scientific (4 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (4 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (3 mentions)
Mirneasy mini kit, by Qiagen (3 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Taqman reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Ddpcr system, by Bio-Rad (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
7900ht thermocycler, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 7900ht thermocycler, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
47 protocols using mirna specific stem loop primer
1
Quantitative RT-PCR Analysis of miRNA
2
Plasma RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In each experiment, RNA was extracted from 200 μl of plasma (two preps obtained from the same human study participant were then combined) using a TRIzol treatment and silica (Ambiom Glass Fiber Microcolumn) binding protocol (http://asuragen.com/wp-content/uploads/2016/05/biomarkers.pdf ). Single-target quantitative RT-PCR (RT-qPCR) was performed using the TaqMan Reverse Transcription Kit and miRNA-specific stem-loop primers (ThermoFisher). The RT step was performed in triplicate, and 2 μl of the original plasma equivalents was present in the final PCR. Placental RNA was used as a “positive control”, and no-template controls were used as a “negative control” for each run. Calibration curves were obtained for each miRNA tested. Quality control of the miRNA preps was performed by testing two ubiquitous miRNAs, namely, miR-16 and miR-27a, known to have low variability in each plasma preparation.
3
Quantification of Circulating miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MicroRNAs were quantified by RT-qPCR as described previously in details [5 (link)]. Following RNA extraction, reverse transcription was performed using Taqman miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (ThermoFisher Scientific). Briefly, a fixed volume of 2.5 µL from the 45 µL RNA eluate was used as input into the RT reaction. In parallel, a 7-point standard curve ranging from 3 × 108 to 3 × 102 copies was reverse-transcribed for each miRNA using synthetic miRNA mimics (ThermoFisher Scientific). Real-time PCR was performed in duplicate using 4.5 µL of the 5-fold diluted RT product combined with 5.5 µL of PCR assay reagents (Taqman miRNA assays, ThermoFisher Scientific) to generate a final PCR volume of 10 µL (Assay ID: hsa-miR-483-5p: 002338; Cel-miR-39: 000200). Assays were run on C1000 Thermal cycler (CFX96 Real Time system, Bio-Rad, Marnes-La-Coquette, France) at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were analyzed with CFX Manager Software version 3.1 (Bio-Rad). Copy number of miR-483-5p in each sample was normalized to C. elegans exogenous spike-in control (Cel-miR-39) using a previously published median normalization procedure, and expressed as normalized copy number per mL of serum [5 (link)].
4
Plasma miRNA Quantification by ddPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasma samples were collected and processed for RNA isolation as previously reported (15 (link)). Absolute levels of miRNAs in plasma were quantified at the droplet digital PCR (ddPCR) system (Bio-Rad Laboratories, Hercules, CA) by using the TaqMan miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Thermo Fisher Scientific, Foster City, CA; cat. No. 4427975; hsa-miR-18a-5p, Assay ID: 002422; hsa-miR-29a, Assay ID: 002112; hsa-miR-20b-5p, Assay ID: 001014) according to the manufacturer's instructions, and following the same experimental protocol described in our previous work (15 (link)).
5
Plasma miRNA Isolation and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
miRNA isolation and qRT-PCR analysis were performed in accordance with the following protocol (Asuragen, Austin, TX). RNA was extracted from 1 ml of plasma using a TRIzol treatment and silica (Ambion Glass Fiber Microcolumn)-binding protocol (http://asuragen.com/wp-content/uploads/2016/05/biomarkers.pdf ). Single-target qRT-PCR was performed using the TaqMan Reverse Transcription Kit and miRNA-specific stem-loop primers (Thermo Fisher). QC of miRNA preps was performed by testing two ubiquitous miRNAs in each plasma prep; all samples with values within two standard deviations of the average value qualified as acceptable for analysis. miRNAs with cycle thresholds (Ct)>37 were excluded from the analysis of each respective sample. The RT step for generation of cDNA from selected miRNAs was performed in triplicate using miRNA-specific primers, and 2-µl plasma equivalents were present in the final PCR. Calibration curves for each miRNA were generated to calculate the miRNA concentration in copy numbers.
6
Quantitative Plasma miRNA Analysis via ddPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative measurements of miRNAs in plasma were carried out using a two-step droplet digital PCR (ddPCR), as previously reported (16 (link)). In the initial step, cDNA was synthesized from 1 μl of extracted RNA using the TaqMan miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Thermo Fisher Scientific, Foster City, CA; cat. No. 4427975; hsa-miR-122-5p, Assay ID: 002245; hsa-miR-1273g-3pg-3p, Assay ID: 475626_mat; has-miR-6126, Assay ID: 475618_mat; hsa-miR-18a-5p, Assay ID: 002422) following manufacturer's instructions. Then, 5 μl of the synthesized cDNA was amplified by mixing 2X ddPCR Supermix for Probes (Bio-Rad Laboratories, Hercules, CA) and 20X TaqMan miRNA PCR primer probe set (Thermo Fisher Scientific, Carlsbad, CA), and quantified by the ddPCR system (Bio-Rad Laboratories, Hercules, CA).
7
Reverse Transcription and qRT-PCR for miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3 µL of RNA was reverse transcribed using the TaqMan miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (ThermoFisher Scientific, Waltham, MA, USA) in a small-scale RT reaction. 0.8 µL of RT product was used for qRT-PCR and was run on QuantStudio 12K using specific TaqMan miRNA assays (ThemoFisher Scientific) [12 (link)].
8
Quantification of miRNA and mRNA Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from cells or tissue was isolated using TRIzol Reagent and was reverse transcribed using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific, Grand Island, NY, USA). cDNA samples were amplified by using SYBR green PCR Master Mix (Thermo Fisher Scientific) with gene specific primers (Supplementary Information Table 2 ). 18S RNA was used as an internal control. RNAs from serum were isolated with mirVana PARIS Kit (Thermo Fisher Scientific) in conjunction with the synthetic spike-in control (cel-miR-39 mimic, QIAGEN, Hilden, Germany). Reverse transcription reactions for miRNA are performed using the TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific) and miRNA-specific stem-loop primers (Thermo Fisher Scientific). The RT products of serum were pre-amplified (Thermo Fisher Scientific) prior to the real-time PCR step to potentially enhance sensitivity. Real-time PCR reactions for miRNA are performed using the TaqMan 2X Universal PCR Master Mix II (Thermo Fisher Scientific) and TaqMan miRNA-specific probe mix (Thermo Fisher Scientific). Relative quantities of miRNAs were calculated by using the 2-ΔΔCt method with U6 snRNA (Thermo Fisher Scientific) as the endogenous control. The assays were listed in Supplementary Information Table 3 .
9
Quantifying miRNA expression from muscle samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
miRNAs were extracted from muscle samples of right and left Biceps femoris of each animal. The mirVana miRNA isolation kit (Ambion, Austin, TX, USA) was used, according to the manufacturer’s instructions, and microRNAs were finally eluted with 100 μL of water and quantified using a Nanodrop spectrophotometer (Labtech, Wilmington Delaware, USA). Reverse Transcription reactions were carried out on 5 ng of miRNAs using the TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) and miRNA-specific stem loop primers for miR-1, miR-133a, miR-206, miR-222, and miR-486 (Applied Biosystems miRNA assays). Real-time PCR reactions were performed at least in duplicate with miRNA-specific primers and Taqman® probes on the CFX96 PCR System (BioRad). Data were normalized using U6 snRNA (RNU6B) as an internal control and differential expression was calculated using the 2-∆∆Ct method. For each miRNA, statistical differences between two groups were analysed by a Mann–Whitney test.
10
Quantifying miRNA Expression in SALS Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 6 SALS and 6 control T75 fibroblast culture flasks using the mirVana isolation kit (Applied Biosystems) according to the manufacture's protocol. TaqMan® Low Density Arrays (TLDAs) were used to measure the expression level of 377 microRNAs (miRNAs) (Applied Biosystems). Briefly, extracted RNA was used in megaplex reverse transcription reactions containing miRNA specific stem loop primers (Applied Biosystems). Following subsequent pre-amplification, the expression of miRNA was measured using Q-PCR with TLDA cards (Human A v3.0). The card data were analysed on an ABI 7900HT Q-PCR system using Sequence Detection System (SDS) software v2.3 according to the manufacturer's recommended conditions. Manual inspection of amplification plots and preliminary data analysis were performed using SDS RQ manager v1.2 and Data Assist software v2.0 respectively (Applied Biosystems). Relative expression of mature miRNAs was calculated using the competitive CT 2ΔΔCt method, with stably expressed miRNAs across all samples, (as identified using NormFinder algorithm [19] (link), acting as controls to normalize the data.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!