Abi 7500 7500 fast real time pcr system

Total RNA extraction was completed from differentiated HFSCs with using a TRIzol (Takara, Japan). To quantity chi-miR-133a-3p and chi-miR-145-5p, we performed reverse transcription with a stem-loop RT-primer and oligo-primers (dT-F17). To assess gene expression, we employed a Takara Prime-Script RT kit accompanied a gDNA eraser (Tokyo, Japan) to reverse-transcribe total RNA into cDNA. All real-time quantitative polymerase chain reaction (RT-q-PCR) reactions were conducted thrice and employed the ABI 7500/7500-Fast real-time PCR system (Applied Biosystems, CA, USA) with a Takara TB Green II Master Mix Reagent Kit (Tokyo, Japan). The 18S RNA (for the chi-miR-133a-3p and chi-miR-145-5p quantification) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the normalization control. The chi-miR-133a-3p, chi-miR-145-5p and genes relative abundances were computed with the 2^−ΔΔCt formula [27 (link),28 (link)]. The miRNA and protein-coding gene primers are summarized in Table 2.

qRT-PCR analysis was used to verify the RNA-seq gene expression pattern. Reactions comprised 500 ng of total RNA, 1 μl of 10 mMdNTPs (Bioline, London, UK) and 1 μl of oligo-dT(20) primers (Invitrogen, Carlsbad, CA, USA), up to a final volume of 11 μl by the addition of RNase-free H₂O. Reactions were performed at 65°C for 5 min, followed by 50°C for 60 min and were then inactivated at 70°C for 15 min.
The qRT-PCR primers were designed based on reference unigene sequences with the Primer Premier5.0 software. The Fast Sybr^RGreen Master Mix (Applied Biosystems,Foster City, CA, USA) was employed, according to the manufacturer’s instructions, in a reaction volume of 10 μl. Real-time PCR was conducted on an ABI 7500/7500 Fast Real-Time PCR system(Applied Biosystems).PCR conditions included initial denaturation for 2 min at 95°C, followed by 40 cycles of denaturation at 95°C for 30 s,hybridizationat 60°Cfor 40 s, and elongation at 68°C for 10 s. The actin2 gene was used as an internal control. The 2^-ΔΔct method was used to calculate the relative level of gene expression, and the B73 sample served as a control. A relative level of gene expression greater than 1 was considered to indicate up-regulation, and less than 1 indicateddown-regulation. All qRT-PCR reactions were performed with the three biological replicates.

Total RNA was extracted from hair follicle stem cells and collected tissues using a TRIzol kit (Takara, Tokyo, Japan). For gene and circCOL1A1 quantification, total RNA was reverse-transcribed into cDNA using the Takara Prime-Script RT kit with gDNA eraser (Tokyo, Japan). For miR-149-5p quantification, stem-loop and oligo-primers (dT-17) were used for reverse-transcription. RT-q-PCR was performed in triplicate on an ABI 7500/7500-Fast Real-Time PCR system (Applied Biosystems, CA, United States) with Takara TB Green II Master Mix Reagent Kit (Tokyo, Japan). GAPDH (for gene and circCOL1A1 quantification) and 18S-rRNA and U6-snRNA (for miR-149-5p quantification) were selected as the internal normalization controls. The relative gene, circCOL1A1 and miR-149-5p expression abundances were calculated using the 2^−ΔΔCt method (Arocho et al., 2006 (link); Adnan et al., 2011 (link)). Primer sequences for RT-PCR and RT-q-PCR are listed in Supplementary Table S3.

Total RNA was extracted using Trizol reagent (Sigma, USA). 2 μg RNA was reverse-transcribed into cDNA using the Revertra ace qPCR RT Kit (TOYOBO, Japan). Real-time PCR was performed in triplicate using AceQ qPCR SYBR green master mix (Vazyme, Nanjing, China) by the ABI 7500/7500 fast Real-Time PCR systems (Applied Biosystems, ThermoFisher, USA). The amount of tRNA was calculated by △△Ct method. The human 18S RNA was used as the internal control. For pre-tRNAs, primers were designed to span the cleavage sites. For mature tRNAs, primers were designed within gene exon, and therefore amplified both pre- and mature mitochondrial tRNAs. The primers were as follows:
mt pre-tRNA^Pro: 5′-GATGGGTGAGTCAATACTTGGG-3′ and 5′-CCATTAGCACCCAAAGCTAAGA-3′; mt pre-tRNA^Val: 5′-AGAGGAGACAAGTCGTAACATGG-3′ and 5′-GGTGCTTTGTGTTAAGCTACACTCTGG-3′; mt pre-tRNA^Ile: 5′-GCGAATTCAATCTCCAGCA-3′ and 5′-GAAATAAGGGGGTTTAAGC-3′; mt tRNA^Pro: 5′-TCAGAGAAAAAGTCTTTAACTCCACC-3′ and 5′-ATAGTTTAAATTAGAATCTTAGCTTTGGGT-3′; mt tRNA^Val: 5′-GTGTAGCTTAACACAAAGCACCC-3′ and 5′-TCAGAGCGGTCAAGTTAAGTTG-3′; mt tRNA^Ile: 5′-TCTTTATACAGACTATTTTC-3′ and 5′-TAGAAATAAGGGGGTTTAAG-3′; 18S RNA: 5′-GCCGCTAGAGGTGAAATTCTT-3′ and 5′-CTTTCGCTCTGGTCCGTCTT-3′.