Mirna specific taqman mirna assay kit

Total miRNA of cells and tissues was extracted using miRNeasy mini Kit (Qiagen) according to the manufacturer's protocol, cDNA was synthesized from 10ng of total miRNA using miR-618 specific primers (TaqMan miRNA assay, Applied Biosystems) and Taqman miRNA Reverse Transcription Kit (Applied Biosystems). miR-618 expression level was quantified using miRNA-specific TaqMan miRNA assay Kit (Applied Biosystems). Total RNA was extracted using TRIzol (Life Technologies), cDNA was reverse transcribed from 5ug total RNA using HiScript 1st Strand cDNA Synthesis Kit (Vazyme). The gene expression level was quantified using AceQ qPCR SYBR Green Master Mix (Vazyme) following the manufacturer's instructions. Real-time RT-PCR was performed using the Applied Biosystems 7500 Sequence Detection System. RUN24 was used as an endogenous control for miRNA date normalization, GAPDH was used as an endogenous control for gene normalization. Relative expressions were calculated using a comparative Ct method 19 (link).

Total RNA derived from clinical specimens or cells was extracted using TRIzol Reagent (Beyotime, Shanghai, China), then subjected to reverse transcription into complementary DNA (cDNA) with BeyoRT™ III M-MLV reverse transcriptase (Beyotime) or TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Following qPCR was carried out using SYBR Master Mix (Applied Biosystems) or miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems). Relative expression of genes was assessed using 2^−ΔΔCt method,25 (link) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH, for circ_LDLR, LDLR and RNF38) or U6 (for miR-7) as internal control. Sequences of qPCR primers were: circ_LDLR, 5ʹ-AGTAGCGTGAGGGCTCTGTC-3ʹ (sense) and 5ʹ-CAGCCAACAAGTTGACATCG-3ʹ (anti-sense); LDLR, 5ʹ-GAATCTACTGGTCTGACCTGTCC-3ʹ (sense) and 5ʹ-GGTCCAGTAGATGTTGCTGTGG-3ʹ (anti-sense); miR-7, 5ʹ-TGGAAGACTAGTGATTTTG-3ʹ (sense) and 5ʹ-GAACATGTCTGCGTATCTC-3ʹ (anti-sense); RNF38, 5ʹ-GGTGAGACTTCAGAGCCTGTTC-3ʹ (sense) and 5ʹ-CGCTGTCTCTTAGGACTTGGAC-3ʹ (anti-sense); GAPDH, 5ʹ-GTCTCCTCTGACTTCAACAGCG-3ʹ (sense) and 5ʹ-ACCACCCTGTTGCTGTAGCCAA-3ʹ (anti-sense); U6, 5ʹ-CTCGCTTCGGCAGCACAT-3ʹ (sense) and 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ (anti-sense).

For miRNA quantification, total RNA including microRNAs was extracted from culture cells and patient samples using the mirVana miRNA Isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions, and then cDNA was synthesized from 5 ng of total RNA using the Taqman^® miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The expression levels of miR-20a were quantified using the miRNA-specific TaqMan^® MiRNA Assay kit (Applied Biosystems) by the 7500 Sequence Detection system (Applied Biosystems). The relative miR-20a expression levels after normalization to U6 small nuclear RNA were calculated using 2-[(Ct of miR-20a) - (Ct of U6)]. qPCR was performed using an SYBR kit [Qiagen China (Shanghai) Co., Ltd.]. The PCR reaction conditions for all assays were as follows: 95°C for 30 sec, followed by 40 cycles of amplification (95°C for 5 sec, 59°C for 30 sec and 72°C for 30 sec). The primers which were synthesized by a TaqMan^® Gene Expression Assay (Invitrogen Life Technologues) were used as follows: Cyclin D1 (Hs00765553/m1) and p21 (Hs00159357/m1). Expression data were normalized to the geometric mean of GAPDH (Hs02758991/g1) to control the variability in expression levels and calculated as 2-[(Ct of cyclin D1 and p21) - (Ct of GAPDH)].

Total RNA of CRC cells and tissues was extracted using TRIzol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Total RNA (1 µg) was reversely transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Dalian, China) and a TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). qRT-PCR was carried out in the Applied Biosystems 7500 Sequence Detection system using a miRNA-specific TaqMan miRNA Assay Kit (Applied Biosystems) and a SYBR Premix Ex Taq™ Kit (Takara, Shiga, Japan) with the following primers: miR-1296-5p (forward primer: 5'-TTG TTA GGG CCC TGG CTC-3'; reserve primer: 5'-GTG CAG GGT CCG AGG T-3'), SFPQ (forward primer: 5'-ATG TCT CGG GAT CGG TTC CGG A-3'; reserve primer: 5'-CCA ACA AAC AAC CGA CAT CGC TG-3'), U6 (forward primer: 5'-CGC TTC GGC AGC ACA TAT AC-3'; reverse primer: 5'-CAG GGG CCA TGC TAA TCT T-3'), GAPDH (forward primer: 5'-TGC ACC ACC AAC TGC TTA GC-3'; reverse primer: 5'-GGC ATG GAC TGT GGT CAT GAG-3'). The relative expression of miR-1296 and SFPQ mRNA were normalized to U6 small nuclear RNA and GAPDH, respectively, using 2^-△△Ct method.