The method used to obtain total RNA was the same as that used for mRNAs. miRNA expression was evaluated as previously described [11 (link)]. The primers for the amplification of miR-34a and RNU6-2 were obtained from QIAGEN (Hilden, Germany). The expression levels of miR-34a were normalized to those of RNU6-2 in the treated cells.
Rnu6 2
Manufactured by Qiagen
Sourced in Germany
RNU6-2 is a small nuclear RNA (snRNA) molecule that is a component of the U6 small nuclear ribonucleoprotein (snRNP) complex. The U6 snRNP is involved in the spliceosome, which is responsible for the removal of introns from pre-mRNA during the process of gene expression.
Automatically generated - may contain errors
4 protocols using rnu6 2
1
Quantification of miR-34a Expression
2
Validation of miRNA Expression in Ovarian Cancer Cells
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Validation with quantitative real-time PCR (qPCR) was performed using cDNA from non-treated (ethanol control) and CLA t10,c12 treated ovarian cancer cells (A2780). Pre-validated RT2 miRNA qPCR assays were purchased from Qiagen: hsa-miR-184 (catalog number: MPH00070A-200), hsa-miR-215 (catalog number: MPH00111A-200), hsa-miR-143 (catalog number: MPH01177A-200), and RNU6-2 (catalog number: MPH01653A-200). All reactions were done in triplicate with three biological repeats. RUN6-2 was used as endogenous control. qPCR was performed in iCycler-myIQ5 (Bio-Rad-Hercules, CA) using conditions that have been previously described with RT2 SYBR Green Fluor qPCR Mastermix (Qiagen) [19 (link)]. RNU6-2 was used as endogenous control. Fold-change in expression compared to control was calculated using delta-delta CT method and mean fold-change is reported. For qPCR of ER stress markers, we followed the same protocol as published in our earlier study [21 (link)]. S27 was used as the housekeeping gene for normalization of the ER stress markers.
3
Quantitative Analysis of miRNA Profiles
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Quantitative PCR (qPCR) analyses were conducted using the QuantiTect SYBR Green PCR System (Qiagen) with a CFX96 thermal cycler (Bio‐Rad, Hercules, CA, USA) as described in our previous study [20 ] with slight modifications. The PCR program was 15 min at 95 °C, followed by 45 cycles of 15 s at 94 °C, 30 s at 55 °C, and 30 s at 70 °C. A miScript Primer Assay (Qiagen) was used to detect miR‐16‐5p, miR‐22‐3p, miR‐30d‐5p, miR‐34a‐5p, miR‐99a‐5p, miR‐125b‐5p, miR‐130a‐3p, miR‐214‐3p, and miR‐451a. The relative expression levels of miRNAs are shown as the ratios of ‘starting quantity’ to ‘protein content in exosome fraction’ and then normalized with values at D0. For qPCR of intracellular miRNA detection, RNU6‐2 (Qiagen) was used as an internal control RNA. The relative expression levels of target miRNAs to those of RNU6‐2 at D12 were normalized to the values at D0. Comparisons between groups were conducted by Student's t‐test. P values of < 0.05 were considered statistically significant.
4
Profiling miRNA Expression in Fibrosis
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Cells were cultured on 35-mm culture dishes and treated with TGF-b1 (10 ng/mL) for 72 h or BLM (0.4 mM) for 144 h. miRNA was extracted from the cells with the miRNeasy Mini Kit. Microarray analysis of miRNAs was performed using 3D-Gene ® system (TORAY, Tokyo, Japan).
miRNA Real-time PCR Analysis Cells were cultured on 35-mm culture dishes and treated with TGF-b1 (10 ng/mL) for 72 h, or BLM (0.4 mM) and MTX (0.1 mM) for 144 h. miRNA was extracted from the cells with miRNeasy Mini Kit and was reverse transcribed into cDNA using miScript Reverse Transcription Kit. Real-time PCR was performed using miScript SYBR Green PCR Kit, according to the manufacturer's instructions. The PCR conditions were as follows: initial denaturation for 1 cycle of 15 min at 95 C, followed by specified cycles of 15 s at 94 C (denaturation), 30 s at 55 C (annealing), and 30 s at 70 C (extension). A melting curve was obtained after the reaction to confirm the single product. The primers for rno-miR-34a-5p, rno-miR-222-3p, rno-miR-200a-3p, and RNU6-2 were purchased from QIAGEN. The expression level of miRNA was normalized as to that of RNU6-2.
miRNA Real-time PCR Analysis Cells were cultured on 35-mm culture dishes and treated with TGF-b1 (10 ng/mL) for 72 h, or BLM (0.4 mM) and MTX (0.1 mM) for 144 h. miRNA was extracted from the cells with miRNeasy Mini Kit and was reverse transcribed into cDNA using miScript Reverse Transcription Kit. Real-time PCR was performed using miScript SYBR Green PCR Kit, according to the manufacturer's instructions. The PCR conditions were as follows: initial denaturation for 1 cycle of 15 min at 95 C, followed by specified cycles of 15 s at 94 C (denaturation), 30 s at 55 C (annealing), and 30 s at 70 C (extension). A melting curve was obtained after the reaction to confirm the single product. The primers for rno-miR-34a-5p, rno-miR-222-3p, rno-miR-200a-3p, and RNU6-2 were purchased from QIAGEN. The expression level of miRNA was normalized as to that of RNU6-2.
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