Briefly, the total cellular RNA was isolated using PureLinkr® RNA Mini Kit (Ambion, USA), reversely transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA), and Quantitative-PCR (TaKaRa Biotechnology Co., Ltd., Japan) analysis according to the manufacturers' instructions. The forward primer for Vimentin was 5′-CCGACACTCCTACAAGATTTAGA-3′, and the reverse primer was 5′-CAAAGATTTATTGAAGGAGAACC-3′. The forward primer for β-actin was 5′- AGCGAGCATCCCCCAAAGTT-3′, and the reverse primer was 5′-CAAAGATTTATTGAAGGAGAACC-3′.
Purelinkr rna mini kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PureLinkr® RNA Mini Kit is a laboratory product designed for the rapid and efficient extraction and purification of total RNA from a variety of sample types. The kit utilizes a silica-based membrane to selectively bind RNA, allowing for the removal of contaminants and inhibitors. The extracted RNA can be used in various downstream applications, such as gene expression analysis, RT-PCR, and RNA sequencing.
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4 protocols using purelinkr rna mini kit
1
Quantitative PCR of Vimentin and β-Actin
2
miRNA Isolation and Quantification
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Total RNA isolation from serum or tissues was performed according to the manufacturer instructions for the PureLinkR RNA Mini Kit (Ambion, Austin, TX, USA) as previously described [31 (link), 32 (link)]. The cDNA derived from the total RNA was prepared using a TaqMan miRNA Reverse Transcription (RT) kit (ABI, Foster City, CA, USA) and miRNA-specific stem-loop primers (part of the TaqMan miRNA assay kit; ABI) as previously described [23 (link), 33 (link)]. For the real-time PCR of the miRNA, we used individual miRNA-specific primers (part of the TaqMan miRNA assay kit; ABI) with the StepOne Real-Time PCR System (ABI) according to the manufacturer’s protocol. Each miRNA was assayed in triplicate and data are presented as median values with the standard deviation. The relative expression levels for each miRNA were normalized using by endogenous and exogenous controls that are miR-16 and cel-miR-39. SDS 2.1 real-time PCR data analysis software (ABI) was used for the data analysis.
3
Transient Transfection and RT-PCR Analysis
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The wild-type and mutant minigene constructs were transiently transfected into HEK293 cells using the promofectine transfection reagent, according to manufacturer's instructions (PromoKine, Heidelberg, Germany). Cells were collected 24 hr post-transfection. Total RNA was extracted using the PureLink R RNA Mini Kit (Ambion; Life Technologies, Saint Aubin, France), according to the manufacturer's instructions, followed by a DNase treatment with the DNA-free Kit (Ambion; Life Technologies). The RT-PCR reactions were performed using the SuperScript OneStep RT-PCR with Platinium Taq kit (Life Technologies), according to the manufacturer's instructions, with 500 ng RNA as a template in a 50-μl reaction volume. Reactions were performed using 300 nM of forward pCAS-KO1F (5 -TGACGTCGCCGCCCATCAC-3 ) and reverse pCAS2R primers (5 -ATTGGTTGTTGAGTTGGTTGTC-3 ) [Gaildrat et al., 2010] . The reverse transcription program had one cycle: 50°C for 30 min. The PCR program had 35 cycles of amplification of 96°C for 45 sec, 50°C for 45 sec, and 72°C for 1 min. RT-PCR products were separated by electrophoresis on 2% agarose 1000 (Invitrogen, Saint Aubin, France) gels containing ethidium bromide and visualized by exposure to nonsaturating ultraviolet light.
4
Quantitative RNA Expression Analysis
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Total cellular RNA was isolated using the PureLink ® R RNA Mini Kit (Ambion, Foster City, CA), reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA), and analyzed by quantitative reverse transcription PCR (TaKaRa Biotechnology Co., Ltd., Shiga, Japan) according to the manufacturers' instructions. The primer sequences are listed in Table 1.
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