miR-140-5p and miR-NC was obtained from Origene Technologies, Inc. The mature-miR-140-5p and miR-NC sequence was cloned into pCDHCMV-MCS-EF1-coGFP constructs (Invitrogen; Thermo Fisher Scientific, Inc.). For STAT3 overexpression, the coding sequences of STAT3 were amplified from cDNA isolated from RA FLS using a PCR kit (AP111-11; TransGen Biotech Co., Ltd.). The thermocycling conditions were as follows: 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 30 sec and 72˚C for 30 sec. Sequences for primers were as follows: STAT3 forward, 5'-GACTTAGTCCCAGGTACT-3' and reverse, TTCAACTGACCTAGGACGTGGTCG-3'. The product was inserted into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate STAT3 expression vectors pcDNA3.1-SOX11. Cells were transfected with pCDHCMV-MCS-EF1-coGFP-miR-140-5p and/or pcDNA3.1-SOX11 using Lipofectamine® RNAiMAX/2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and then collected at 48 h after transfection. All constructions were confirmed using plasmid DNA sequencing using the dideoxy chain-termination (Sanger) method with Applied Biosystems 3730XL (Genescript).
Mir 140 5p
Manufactured by Thermo Fisher Scientific
Sourced in United States
MiR‐140‐5p is a microRNA (miRNA) that functions in gene expression regulation. It is a short, non-coding RNA molecule that plays a role in post-transcriptional regulation of target messenger RNAs (mRNAs). The core function of MiR‐140‐5p is to bind to specific mRNA sequences, leading to either translational repression or mRNA degradation.
Automatically generated - may contain errors
Lab products found in correlation
Mir nc, by OriGene (1 mentions)
Mir nc, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Rnu48, by Thermo Fisher Scientific (1 mentions)
Taqman probes and primers, by Thermo Fisher Scientific (1 mentions)
Mir 140 3p, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mir 342 3p, by Thermo Fisher Scientific (1 mentions)
Mir 223 3p, by Thermo Fisher Scientific (1 mentions)
3 protocols using mir 140 5p
1
miR-140-5p and STAT3 Overexpression in RA FLS
2
Quantifying PLOD1, miR-140-5p, and miR-140-3p
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TaqMan probes and primers specific to PLOD1 (P/N: Hs00609363_m1; Applied Biosystems), which are assay‐on‐demand gene expression products, were used to analyze PLOD1 expression. miR‐140‐5p (P/N:001187; Applied Biosystems) and miR‐140‐3p (P/N:002234; Applied Biosystems) expression was analyzed by qRT‐PCR. mRNA and miRNA expression levels were normalized to those of GUSB (P/N: Hs99999908_m1; Applied Biosystems) and RNU48 (assay ID: 001006; Applied Biosystems). PCR quantification was performed as described previously (Yamada et al., 2018d ).
3
miRNA Expression Profiling by RT-qPCR
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miRNAs were determined using two-step RT-qPCR with RT-primer specific assay in combination with TaqMan probes: miR-143-5p (Assay ID: 463509_mat), miR-140-5p (Assay ID: 001187) miR-148a-5p (Assay ID: 473012_mat), miR-450a-5p (Assay ID: 462729_mat), miR-21-5p (Assay ID: 000397), miR-146b-5p (Assay ID: 002755), miR-342-3p (Assay ID: 002260), and miR-223-3p (Assay ID: 07896_mat) (Applied Biosystems; CA, USA). Each RT-reaction used 1.5 µL from the 14 µL eluted RNA using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; CA, USA). The RT reaction program and PCR cycling conditions were as we previously reported (Brianza-Padilla et al., 2016 (link)). miRNAs relative concentrations were normalized with Ct values of cel-miR-39, and values were calculated using 2−ΔΔCt and 2−ΔCt formulas. All Ct values for cel-miR-39 ranged from 20 to 22 cycles both for total plasma and for EVs RNA isolations.
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