Total RNA from cells or tissues was extracted with the TRIzol reagent (Invitrogen) and then reversely transcribed into cDNA using the PrimeScript RT kit (Takara, Kusatsu, Japan). For miRNA detection, the RNA was reverse transcribed into cDNA using the miRcute Plus miRNA first-strand cDNA synthesis kit (TianGen, Beijing, China). miRNA in the medium (350 μL) and EVs (100 μg) was extracted with the help of the mirVana PARIS Kit (Ambion, Austin, TX). An exogenous reference cel-miR-39 (1 pmol/sample; TianGen) was added. RT-qPCR for mRNA was performed using the SYBR Premix Ex Taq Reagent Kit (Takara) and ABI StepOne real-time PCR system (Applied Biosystems, Foster City, CA). RT-qPCR for miRNA was processed utilizing the miRcute Plus miRNA qPCR Detection Kit (TianGen). β-Actin was used as a normalizer for mRNA while U6 for miRNA. In addition, miRNA expression in culture media and EVs was normalized to the exogenous reference cel-miR-39. The relative expression of target genes was calculated by the 2ΔΔCT method. Primer sequences are described in Supplementary Table 1 .
Cel mir 39
Manufactured by Tiangen Biotech
Sourced in China
Cel-miR-39 is a synthetic microRNA (miRNA) derived from the nematode Caenorhabditis elegans. It is commonly used as a spike-in control for miRNA detection and quantification in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Mircute plus mirna qpcr detection kit, by Tiangen Biotech (3 mentions)
Mirvana paris kit, by Thermo Fisher Scientific (3 mentions)
Mircute microrna isolation kit, by Tiangen Biotech (3 mentions)
First strand cdna synthesis kit, by Tiangen Biotech (3 mentions)
Mircute plus mirna first strand cdna synthesis kit, by Tiangen Biotech (2 mentions)
Abi stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq reagent kit, by Takara Bio (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Microrna reverse transcription kit, by EZBioscience (1 mentions)
Mircute plus mirna qpcr kit, by Tiangen Biotech (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
8 protocols using cel mir 39
1
Quantitative Analysis of miRNA Expression
2
microRNA Isolation and Quantification
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2 μl of 25 fmol cel-miR-39 (Tiangen, Beijing, China) was added to 200 μl of serum samples as external reference. Total RNA was isolated simultaneously using the miRcute microRNA Isolation Kit (Tiangen, Beijing, China) abiding by the manufacturer’s protocol [23 (link)]. The optical density of the extracted total RNA was determined at 260 and 280 nm on a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE, USA) to assess for concentrations and purities.
The extracted microRNA was polyadenylated with poly (A) polymerase in a 20-μl volume, and 6 μl of the poly (A) reaction solution was reversely transcribed to cDNA in another 20 μl with miRcute microRNA The First-strand cDNA Synthesis Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. All procedures were carried out in triplicates to remove outliers.
The extracted microRNA was polyadenylated with poly (A) polymerase in a 20-μl volume, and 6 μl of the poly (A) reaction solution was reversely transcribed to cDNA in another 20 μl with miRcute microRNA The First-strand cDNA Synthesis Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. All procedures were carried out in triplicates to remove outliers.
3
Serum miRNA Isolation and Quantification
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A total of 200 μL of the serum samples was spiked with 2 μL of 25 fmol synthetic cel‐miR‐39 (Tiangen, Beijing, China) as the external reference. Total RNA from the serum samples was isolated simultaneously using the miRcute microRNA Isolation Kit from Tiangen following the manufacturer's instructions.13 To determine the concentrations and purities, the optical density of the extracted total RNA was assessed at 260 and 280 nm on a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE, USA).
The extracted microRNA was polyadenylated by poly (A) polymerase in a 20‐μL volume, and 6 μL of the poly (A) reaction solution was reverse‐transcribed to cDNA in another 20 μL using miRcute microRNA The First‐strand cDNA Synthesis Kit from Tiangen was used according to the manufacturer's instructions. Reverse transcription was performed in triplicate to remove outliers.
The extracted microRNA was polyadenylated by poly (A) polymerase in a 20‐μL volume, and 6 μL of the poly (A) reaction solution was reverse‐transcribed to cDNA in another 20 μL using miRcute microRNA The First‐strand cDNA Synthesis Kit from Tiangen was used according to the manufacturer's instructions. Reverse transcription was performed in triplicate to remove outliers.
4
Comprehensive miRNA and mRNA Profiling
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Total RNA was extracted from cultured cells or tissues using the TRIzol reagent (Invitrogen). For mRNA detection, reverse transcription kit (RR047A, Takara, Japan) was adopted to obtain cDNA. MicroRNAs in medium (350 μL) and EVs (100 μg) were extracted using mirVana PARIS Kit (Ambion, Naugatuck, CT, USA), with cel-miR-39 (1 pmol per sample; TianGen, Beijing, China) as an exogenous reference. For miRNA detection, microRNA Reverse Transcription Kit (EZB-miRT2-S, EZBioscience, USA) was applied for generating cDNA. microRNA-cDNAs were quantitatively amplified utilizing miRcute Plus miRNA qPCR Detection Kit (TianGen) with U6-cDNA as an internal reference. GAPDH was regarded as the internal reference of mRNA. The expression of each gene or isoform was quantified using the comparative threshold method with the formula of 2−ΔΔCt. The primer sequences of quantitative amplification were listed below Table S3 [48 (link)].
5
Extracellular Vesicle miRNA Analysis in Diabetes
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First-morning urine from all of the DM and DN patients (see additional information for inclusion and exclusion criteria) was collected. Extracellular vesicles from urine and cell culture medium were subjected to differential centrifugation as previously described22 . The pellets were suspended in 100 μl of PBS and stored at −80 °C. The presence of extracellular vesicles was verified by transmission electron microscopy as previously described22 . miRNA was isolated from extracellular vesicles using TRIzol. Notably, 25 fmol cel-miR-39 (TIANGEN, Beijing, China) was added to extracellular vesicle samples after a 5 min incubation in TRIzol as previously described22 . miRNA expression values were normalized to those of cel-miR-39.
This study was approved by the ethics committee of Nanfang Hospital, Southern Medical University, and conducted in accordance with the guidelines of the ethical management. All of the patients provided signed informed consent prior to participating in the study.
This study was approved by the ethics committee of Nanfang Hospital, Southern Medical University, and conducted in accordance with the guidelines of the ethical management. All of the patients provided signed informed consent prior to participating in the study.
6
Serum microRNA Isolation and cDNA Synthesis
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200 μl of the serum samples was spiked with 2 μl of 25 fmol synthetic cel-miR-39 (Tiangen, Beijing, China) as the external reference. Total RNA enriched for small RNAs was isolated simultaneously from the serum with the miRcute microRNA Isolation Kit (Tiangen, Beijing, China) according to the modified manufacturer’s protocol [19 (link)]. To determine the purities and concentrations, we utilized a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE, USA) to assess the optical density of the extracted RNA at 260 and 280 nm.
The extracted microRNA was polyadenylated by 20 μl of the poly (A) polymerase. 6 μl of the poly (A) reaction solution was reverse transcribed to cDNA in another 20 μl with miRcute microRNA The First-strand cDNA Synthesis Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. Reverse transcription was run in triplicate.
The extracted microRNA was polyadenylated by 20 μl of the poly (A) polymerase. 6 μl of the poly (A) reaction solution was reverse transcribed to cDNA in another 20 μl with miRcute microRNA The First-strand cDNA Synthesis Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. Reverse transcription was run in triplicate.
7
Quantitative miRNA Expression Analysis
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The Trizol reagent (16096020, Thermo Fisher Scientific) was employed for extracting the total RNA content from tissues and cells. cDNA of miRNA was synthesized from the total RNA of cells and tissues using the miRcute Plus miRNA First-Strand cDNA Synthesis Kits (TIANGEN Biotechnology Co. Ltd, Beijing, China). Next, the synthesized exogenous reference cel-miR-39 (1 pmol per sample, TIANGEN Biotechnology Co. Ltd) was added to 350 μL medium or 100 μg EVs in advance. The miRNA was extracted from these samples with a mirVana PARIS kit (Ambion Company, Austin, TX). Real-time PCR of miRNA was carried out using a miRcute Plus miRNA qPCR Kit (TIANGEN Biotechnology Co. Ltd). In cell and tissue lysates, miRNA expression was standardized to U6. Meanwhile, miRNA expression in the medium and EVs were standardized relative to the exogenous reference cel-miR-39. miRNA universal reverse primers were provided by the miRcute Plus miRNA First-Strand cDNA Synthesis kit. The remaining primers were synthesized by Sangon (Shanghai, China). The primers are manifested in Supplementary Table 2 . The 2−ΔΔCT method was employed for analysis.
8
Quantitative mRNA and miRNA Analysis
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Total RNA extracted from tissues or cells using Trizol (Thermo Fisher Scienti c, New York, USA) was taken to perform reverse transcription to obtain cDNA or miRNA cDNA with a reverse transcription kit (Fermentas Inc., Ontario, CA, USA) or miRcute Plus miRNA First-Strand cDNA Synthesis Kit (TIANGEN, China). Synthetic exogenous internal reference cel-miR-39 (1 pmol per sample; TIANGEN, China) was supplemented to culture medium (350 μL) or EVs (100 μg) prior to the experiment. Subsequently, miRNA was isolated from these samples using the mirVana PARIS kit (Ambion, USA). mRNA RT-qPCR was completed with SYBR® Premix Ex Taq (Takara, Japan) on the ABI StepOne real-time PCR system (Applied Biosystems, USA). Meanwhile, miRcute Plus miRNA qPCR detection kit (TIANGEN, China) was adopted in miRNA RT-qPCR. GAPDH was used as the internal control in measuring mRNA expression in cells and tissues. U6 was used as the internal control in measuring miRNA expression. In addition, miRNA level was normalized relative to exogenouscel-miR-39. The miRNA universal reverse primer was obtained from miRcute Plus miRNA qPCR detection kit (TIANGEN, China), and other primers were provided by Shanghai Sangon (see Table 1 for primer sequences). All experiments were processed in triplicates and the results were analyzed following the 2 (-ΔΔCT) method.
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