RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, Inc.) according to manufacturer's protocol for preservation of small RNAs. RNA for miR detection was reverse transcribed using the Universal cDNA Synthesis kit (Exiqon, Inc., Woburn, MA, USA) with 100 ng RNA, under the following conditions: 60 min at 42°C, followed by 5 min at 95°C and hold at 4°C. qPCR was performed on the cDNA using the ExiLENT SYBR Green Master mix (Exiqon, Inc.) according to the manufacturer's protocol. The StepOne Plus Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used for detection and quantitation. The following cycling conditions were employed for qPCR: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 1 min. Primers for the following were used: small nucleolar RNA U66 (snoR-U66) (product no. 203905), miR-96 (product no. 204417), miR-182 (product no. 206070) and miR-183 (product no. 206030; Exiqon, Inc.). Relative quantity was calculated by the ΔΔCq method (31 (link)) and normalized to snoR-U66.
Mir 182
Manufactured by Qiagen
Sourced in Denmark
MiR-182 is a laboratory equipment product manufactured by Qiagen. It is designed for the detection and analysis of miR-182, a specific microRNA. The core function of MiR-182 is to provide researchers with the necessary tools to study the expression and regulation of miR-182 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Universal cdna synthesis kit, by Qiagen (1 mentions)
Exilent sybr green master mix, by Qiagen (1 mentions)
Mir 127 3p, by Qiagen (1 mentions)
Sybr green master mix, by Qiagen (1 mentions)
Steponeplus real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Mir 21, by Qiagen (1 mentions)
Mircury lnatm synthesis kit 2, by Qiagen (1 mentions)
Mircury lna exilent sybr green, by Qiagen (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
3 protocols using mir 182
1
miRNA Expression Analysis by qPCR
2
Quantitative Analysis of Bovine Follicular Fluid miRNAs
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Based on their enrichment in follicular fluid and blood plasma from hyperstimulated heifers, 11 candidate miRNAs were selected for further study. Moreover, high merit was given to those miRNAs which has sequence similarities between human and bovine during the selection of candidate miRNAs. Primer sets of individual qRT-PCR assays for miRNAs: miR-212, miR-182, let-7 g, miR-100, miR-877, miR-200c, miR-221, miR-103, miR-134, miR-147 and miR-127-3p were obtained from Exiqon (Vedbaek, Denmark). Reverse transcription reaction products were combined with SYBR Green master mix (Exiqon, Vedbaek, Denmark) and loaded into the 96-well plates preloaded with individual miRNA primers. Quantitative PCR was run in a Step one plus real time PCR instrument (Applied Biosystems). Relative expression of each miRNA was analysed using a comparative Ct (2-ΔΔCT) method and global normalization strategy was employed to normalize the data.
3
Quantifying miRNA Expression in Cell Lines
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To determine the expression of miRNAs (miR-21, miR-182, miRNA-7 and miR-SNORD 44) (Exiqon) in control non-treated cells/PRP treated cells, TRIZOL Reagent (Life Technologies) was used according to the manufacturer’s recommendations. Reverse transcription from miRs was performed using the miRCURY LNATM Synthesis kit II (Exiqon) following the manufacturer’s protocol. qPCR was performed using miRCURY LNA TM EXILENT SYBR Green (Exiqon) in a CFX96 real-time PCR detection system (Bio-Rad). Each reaction was performed in triplicate and the comparative threshold cycle (Ct) method was used to calculate the amplification factor. Human Snord 44 was used as housekeeping.
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