RNA was extracted from BMMs using a RNA isolation kit (Takara) according to the manufacturer’s instructions. A quantitative RT-PCR analysis was performed using the SYBR Green PCR Master Mix (Takara Bio). miR-149-5p in EVs from BMMs supernatant or serum of mice was extracted using the miRNeasy Serum/Plasma kit (QIAGEN). The expression of miR-149-5p was detected with the miRNA qRT-PCR Detection Kit (GeneCopoeia). Levels of each mRNA or miRNA, respectively, were normalized to the GAPDH or U6 levels. The miR-149-5p level of EVs was compared with spiked-in ce-miR-39 to normalize miRNA expression, which was used as the reference by using the miRNeasy Serum/Plasma Spike-In Control kit (QIAGEN). Each experiment was performed at least 3 times. The 2−ΔΔCT method was used to quantify expression of the genes of interest. The primer sequences used in this study are listed in Supplemental Table 1 .
Mirneasy serum plasma spike in control kit
Manufactured by Qiagen
Sourced in United States
The MiRNeasy Serum/Plasma Spike-In Control kit is a laboratory tool designed to aid in the analysis of microRNA (miRNA) levels in serum and plasma samples. The kit includes synthetic miRNA that can be spiked into samples as an internal control, allowing for the normalization of miRNA quantification results.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy serum plasma kit, by Qiagen (6 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Rna isolation kit, by Takara Bio (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Mirna qrt pcr detection kit, by GeneCopoeia (1 mentions)
Hiscript 2 q rt supermix for qpcr, by Vazyme (1 mentions)
All in one mirna rt qpcr detection kit, by GeneCopoeia (1 mentions)
Synthetic c elegans mir 39, by Qiagen (1 mentions)
Proteinase k, by Zymo Research (1 mentions)
7 protocols using mirneasy serum plasma spike in control kit
1
Quantitative Analysis of miRNA-149-5p in BMM-Derived EVs
2
Vitreous miRNA Isolation and Profiling
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Fifty μL of each individual vitreous sample from each group were pooled to a total of 1 mL/group (pooled analysis). The miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany; Cat No. 217184) was used for the isolation of cell-free total RNA including miRNA from the vitreous, according to the manufacturer’s instructions. The miRNeasy Serum/Plasma Spike-In Control Kit (Qiagen, Cat No. 219610) was used during the extraction protocol. The Kit includes a C. elegans miRNA-39-mimic (Ce), which serves as an isolation efficiency control and internal normalization control for the miRNA profiler assays. The presence of miRNA in each pooled vitreous group was confirmed by running control reactions as described in “Pooled analysis”.
3
Exosomal miR-206-3p Quantification Protocol
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Total RNA was extracted from cells by Trizol reagent according to the manufacturer's instructions. The mRNA was employed to generate cDNA through HiScript® II Q RT SuperMix for qPCR (Vazyme, China) according to the manufacturer's recommendations. Exosomal miR-206-3p was extracted by using miRNeasy Serum/Plasma kit (QIAGEN, USA). miR-206-3p expression was detected through the All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, USA). Levels of each miRNA or mRNA were normalized to the U6 or GAPDH levels. The primers used are listed in Supplemental Table S1 . To normalize miRNA expression, the miR-206-3p levels of exosomes were compared to spiked-in ce-miR-39, which was used as the reference by using the miRNeasy Serum/Plasma Spike-In Control kit (QIAGEN, USA).
Each experiment was performed three times. Data were quantified using the 2-∆∆CT method.
Each experiment was performed three times. Data were quantified using the 2-∆∆CT method.
4
Quantification of miRNA Expression in Extracellular Vesicles
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Total RNA was extracted from cells using Trizol reagent and reverse transcribed using the PrimeScript RT Reagent Kit (Takara Bio, Kusatsu, Japan). miRNAs of purified apoVs were extracted using the miRNeasy Serum/Plasma kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. miRNAs expression were detected by the All‐in‐One miRNA RT‐qPCR Detection Kit (GeneCopoeia, Rockville, MD). The levels of each mRNA or miRNA were normalized to the GAPDH or U6 levels, respectively, while the miRNAs levels from apoVs were compared to the levels of spiked‐in ce‐miR‐39, which was applied as the reference using the miRNeasy Serum/Plasma Spike‐In Control kit (QIAGEN, Valencia, CA). Each experiment was performed in triplicate. The 2−ΔΔCT method was used to quantify expression of the genes of interest. The primer sequences used in this study are listed in Table S8 (Supporting Information).
5
Exosomal miR-31a-5p Expression Analysis
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Total RNA was extracted from cells using Trizol reagent according to the manufacturer's instructions. Reverse transcription was performed with 1 μg of total RNA in a final volume of 20 μl using the PrimeScript RT reagent kit (Takara Bio, Shiga, Japan) according to the manufacturer's recommendations. miR‐31a‐5p of purified exosomes was extracted using the miRNeasy Serum/Plasma kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. Expression of miR‐31a‐5p was detected by the All‐in‐One miRNA qRT‐PCR Detection Kit (GeneCopoeia, Rockville, MD, USA). The levels of each mRNA or miRNA were normalized to the β‐actin or U6 levels, respectively, while the exosomal miRNA levels were compared to the levels of spiked‐in ce‐miR‐39, which was applied as the reference using the miRNeasy Serum/Plasma Spike‐In Control kit (QIAGEN, Valencia, CA, USA). Each experiment was performed in triplicate. The primer sequences used in this study are listed in Table S1 . The 2−ΔΔCT method was used to quantify expression of the genes of interest.
6
Quantification of miRNA in extracellular vesicles
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Total RNA was extracted from cells using Trizol reagent according to the manufacturer's instructions. Reverse transcription was performed with 1 μg of total RNA in a final volume of 20 μL using the PrimeScript RT reagent kit (Takara Bio, Shiga, Japan) according to the manufacturer's recommendations. The levels of each mRNA or miRNA were normalized to the GAPDH or U6 levels, respectively.
miR-143/145 in EVs from human samples or mice BMSCs supernatant were extracted using the miRNeasy Serum/Plasma kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. miRNA in EVs were purified and detected by the All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA). To normalize miRNA expression, miR-143/145 levels were compared to spiked-in ce-miR-39, which was applied as the reference using the miRNeasy Serum/Plasma Spike-In Control kit (QIAGEN, Valencia, CA, USA). Each experiment was performed in triplicate. The primer sequences used in this study are listed inTable S1 . The 2-ΔΔCT method was used to quantify expression of the genes of interest.
miR-143/145 in EVs from human samples or mice BMSCs supernatant were extracted using the miRNeasy Serum/Plasma kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. miRNA in EVs were purified and detected by the All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA). To normalize miRNA expression, miR-143/145 levels were compared to spiked-in ce-miR-39, which was applied as the reference using the miRNeasy Serum/Plasma Spike-In Control kit (QIAGEN, Valencia, CA, USA). Each experiment was performed in triplicate. The primer sequences used in this study are listed in
7
Optimized cfRNA Extraction from Serum
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RNA was extracted with a Quick-cfRNATM Serum & Plasma Kit (Zymo research, Irvine, CA, USA) combining with miRNeasy Serum/Plasma Spike-In Control kit (Qiagen, Hilden, German) and 100% pure ethanol (Merck Millipore, Germany), following to the protocol recommended by the manufacturers. Briefly, 600 µL of serum were homogeneously mixed with 600 µL of Quick-cfRNATM reagent Digestion Buffer (Zymo research, Irvine, CA, USA). After adding 20 µL Proteinase K (Zymo research, Irvine, CA, USA) the homogenate was incubated for two hours at 37°C, then 10 µL (4×109 ng/µL) synthetic C. elegans miR-39 (Qiagen, Hilden, German) was spiked-in the samples following the adjunction of denaturing solution as an external reference for the normalization of miRNA level. Finally, elution was achieved with a spin column and 100% ethanol and eluted RNA was collected RNase-free water.
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