Sybr primescript mirna rt pcr kit

Total RNA was extracted from cultured cells, OSCC clinical tissues, and mice tongue tissues using TRIzol Reagent (Invitrogen, CA, USA). The miRNA was extracted from cultured cells using miRNAiso for small RNA reagent (TaKaRa, Dalian, China). The RNA samples were then reverse-transcribed into cDNA with the PrimeScript RT Master Mix (TaKaRa, Dalian, China) and the SYBR PrimeScript miRNA RT-PCR Kit (TaKaRa, Dalian, China). Real-time PCR was performed with the SYBR Premix Ex Taq II and the SYBR PrimeScript miRNA RT-PCR Kit (TaKaRa, Dalian, China), using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The expression of HTT mRNA was quantified with GAPDH mRNA expression as an endogenous control. The levels of miR-146a were quantified with U6 control. Quantification of the relative levels was determined by the ^ΔΔCt method (28 (link)). Primers used for qPCR are listed in Table 2.

Total RNA was extracted from tissues and cells using Trizol reagent (Invitrogen) following the protocols of manufacturer. RNA was reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific, Rockford, IL, USA) or SYBR PrimeScript miRNA RT-PCR Kit (Takara, Dalian, China). The expression levels were detected using SYBR Green Mixture (Takara) or SYBR PrimeScript miRNA RT-PCR Kit (Takara). GAPDH or U6 was detected as internal control. Primers as follows: LINC00460 (forward, 5′-GTGGATGAGAACGAAGGTTACG-3′; reverse, 5′-CTTTCCCACGCTCAGTCTTT-3′), miR-590-3p (forward, 5′-CCAAGCTTCACCCATTCCTAACAGGAC-3′; reverse, 5′-CGGGATCCGTAGGTCAGTTACATGCATC-3′), Raf1 (forward, 5′-GGGAGCTTGGAAGACGATCAG-3′; reverse, 5′-ACACGGATAGTGTTGCTTGTC-3′), GAPDH (forward, 5′-GACTCCACTCACGGCAAATTCA-3′; reverse, 5′-TCGCTCCTGGAAGATGGTGAT-3′), U6 (forward, 5′-CTCGCTTCGGCAGCACATATACT-3′; reverse, 5′-CGCTTCACGAATTTGCGTGT-3′).

Total RNAs from cultured chondrocytes were isolated with Trizol reagent (Invitrogen, CA, USA). MiRNA was extracted from cultured chondrocytes using RNAiso for small RNA reagent (TAKARA, JAPAN). RNA samples were reverse-transcribed into cDNA with the PrimeScript RT Master Mix (total RNA) and the SYBR PrimeScript miRNA RT-PCR Kit (TAKARA, JAPAN). Real-time PCR was performed with gene-specific primers (Table 2), the SYBR Premix Ex Taq II, and the SYBR PrimeScript miRNA RT-PCR Kit (TAKARA, JAPAN), using the ABI StepOnePlus Sequence Detection System v2.1 (Applied Biosystems, Singapore). The expression of PIK3R1, COL2A1, ACAN, ADAMTS-5, and MMP13 mRNA was quantified with GAPDH mRNA expression as endogenous control. The expression of miRNA was quantified with RNU6B control. Quantification of the relative levels was determined by the ΔΔCt method according to previous studies19 (link)50 (link).

To validate the high‐throughput sequencing results, RT‐qPCR on miRNAs and their targets was performed on a 7300 Real‐Time PCR System (Bio‐Rad, Hercules, CA). We chose 12 miRNA‐target pairs, including 12 differentially expressed miRNAs and 20 targets, for the RT‐qPCR analysis. The RNA samples used for RT‐qPCR were the same as for the experiments mentioned above. The reverse transcription reactions for miRNAs and cDNA were carried out using the SYBR^® PrimeScript^™ miRNA RT‐PCR Kit (TaKaRa, Dalian, China) and the Superscript III First‐Strand Synthesis system followed by RNase H treatment (Invitrogen, Carlsbad, CA), respectively. The specific miRNA forward primers were designed based on the sequences of the respective miRNAs, and the primers for the target genes were designed using the online Primer3 program (http://frodo.wi.mit.edu/primer3/). Beta‐tubulin (TUB) was selected as a reference gene (Sang et al., 2013). The gene names, sequences and the primers used for RT‐qPCR analysis are available in File S1. RT‐qPCR reactions for miRNAs and their targets were performed in 96‐well plates using a SYBR^® Premix Ex Taq^™ Kit (TaKaRa, Dalian, China) and SYBR^® PrimeScript^™ miRNA RT‐PCR Kit (TaKaRa, Dalian, China). The amplification procedure and further data analysis were performed according to a previous study (Han et al., 2012). Each PCR reaction was repeated three times independently.

Total RNA was extracted from head kidney tissue samples from GIFT exposed to 96h-LT₅₀ cold stress (LTS group) or to 28 °C (CO group) for 24 h; two samples from each of the six CO and six LTS tanks, using Trizol reagent (Invitrogen, CA, USA). A Mir-X™ miRNA First-Strand Synthesis kit and a SYBR^® PrimeScript^TM miRNA RT-PCR kit (Takara, Dalian, China) were used for the RT reaction and qRT-PCRs of the miRNAs as described previously^{34 (link)}. The relative fold changes in the expression levels of the miRNAs relative to the expression of U6 (used as the reference gene) were calculated by the 2^−ΔΔCt method. The miRNA specific primers were synthesized by Genewiz, Inc. (Genewiz, Suzhou, China).

Bone tissue and cell miRNAs were isolated by miRNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. For serum miRNAs, the method was similar to the previously described4 (link). In briefly, pipetted 200 μL serum sample accurately were added with 600 μL RNAiso for small RNA (Takara, Dalian, China). Cel-miR-39 was spike-in prior to RNA isolation to act as an additional quality control. Then miRNAs were isolated according to the manufacturer’s instructions, followed by reverse transcription with SYBR PrimeScript^TM miRNA RT-PCR Kit (Takara, Dalian, China). qPCR was conducted in a total volume of 20 μL using SYBR Premix Ex Taq^TM Kit (Takara, Dalian, China) and the PCR cycling program included 95 °C for 2 min, 40 cycles of 95 °C for 10 s, 60 °C for 20 s, 72 °C for 20 s, melting curve analysis was carried out at the end of cycling program. The qPCR was performed in triplicate on LightCycler^®96 (Roche, Switzerland).

Total RNA was isolated from the different cell groups using the RNAfast200 Total RNA Extract Kit (Fastgene, Shanghai, China), and 2 μg RNA was reverse transcribed to cDNA by the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, MBI, Lithuania). Quantitative RT-PCR was carried out using the SYBR^® PrimeScriptTM miRNA RT-PCR Kit and SYBR^® Premix Ex TaqTM (TaKaRa Biotechnology, Dalian, China). The Fold change of gene expression was calculated based on the threshold cycle (Ct) as relative to β-actin using a 2-Δ(ΔCt) method. The sequences of PCR primers for target genes are listed as followings. TP63: forward primer, 5’-GGACCAGCAGATTCAGAACGG-3’; reverse primer, 5’-AGGACACG TCGAAACTGTGC-3’; TJP1: forward primer, 5’-CAACATACAGTGACGCTTCACA-3’; reverse primer, 5’- CACTATTGACGTTTCCCCACTC-3’; YWHAE: forward primer, 5’- GATTCGGGAATATCGGCAAATGG-3’; reverse primer, 5’-GCTGGAATGAGGTGTTTG TCC-3’. All primer pairs were synthesized by TaKaRa.