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One step sybr primerscript rt pcr kit

Manufactured by Takara Bio
Sourced in China

The One Step SYBR PrimerScript RT-PCR Kit is a laboratory tool designed for reverse transcription and real-time PCR amplification in a single reaction. The kit includes all the necessary components to perform this two-step process efficiently.

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3 protocols using one step sybr primerscript rt pcr kit

1

Quantitative Analysis of miRNA and Gene Expression

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The total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and mature miR-185 expression was detected in cells using a Hairpin-it miRNAs qPCR kit (Shanghai Genepharma Co., Ltd.). Thermocycling conditions included an initial step at 98°C for 1 min, and 40 cycles at 95°C for 5 sec and at 62–68°C for 15 sec, 72°C for 2 min and final extension at 72°C for 10 min. The expression of small nucleolar RNU6B was used as an endogenous control. SOX9, β-catenin and c-Myc expression was measured using a SYBR green qPCR assay (One Step SYBR PrimerScript RT-PCR Kit, Takara Biotechnology Co., Ltd., Dalian, China). Reverse transcription and amplification were performed at 42°C for 15 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec using an ABI Prism 7900 HT (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primer sequences were used for the PCR: RNU6B, reverse 5′-AACGCTTCACGAATTTGCGT-3, forward 5′-CTCGCTTCGGCAGCACA-3; SOX9, forward 5′-AGCGAACGCACATCAAGAC-3′, reverse 5′-CTGTAGGCGATCTGTTGGGG-3′; β-catenin, forward 5′-AAAGCGGCTGTTAGTCACTGG-3′, reverse 5′-CGAGTCATTGCATACTGTCCAT-3′; c-Myc, forward 5′-GGCTCCTGGCAAAAGGTCA-3′, reverse 5′-CTGCGTAGTTGTGCTGATGT-3′; GAPDH, forward 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′. Data were analysed using the 2−ΔΔCq method (21 (link)).
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2

Plasma KCNQ1OT1 Detection in TIA

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Blood samples were centrifuged to obtain the supernatant plasma at the first onset of TIA. The TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, United States) was utilized to dissociate the total RNAs from patients’ plasma. Then, the plasma KCNQ1OT1 level was detected via real-time quantitative polymerase chain reaction (qPCR) using the One-Step SYBR Primer Script RT-PCR Kit (Takara Bio, Inc., Japan). The total volume of the reaction system is 20 μl. Melt curves were analyzed to determine the specificity of the amplified products. Primer sequences for KCNQ1OT1 detection were designed as follows: forward 5′-TGC​AGA​AGA​CAG​GAC​ACT​GG-3′ and reverse 5′-CTT​TGG​TGG​GAA​AGG​ACA​GA-3’. GAPDH was regarded as the endogenous reference with the following sequences of primers: forward 5′- TGC​ACC​ACC​AAC​TGC​TTA​GC-3′ and reverse 5′-GGC​ATG​CAC​TGT​GGT​CAT​GAG-3’.
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3

Gene Expression Analysis of HOTAIR and miR-148b-3p

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The expression levels of HOTAIR and miR-148b-3p were detected by qRT-PCR. All qRT-PCR analyses were conducted by means of a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Total RNA was extracted from ECs, GECs and transfected cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA) by the manufacturer's instructions. The RNA concentration and quality were determined by the NanoDrop Spectrophotometer (ND-100; NanoDrop, Wilmington, DE). Primers of HOTAIR were as follows: forward: 5′-GGT AGA AAA AGC AAC CAC GAA GC-3′; reverse: 5′-ACA TAA ACC TCT GTC TGT GAG TGC C-3′. Quantitative RT-PCR of HOTAIR was conducted using One Step SYBR® Primer Script™ RT-PCR Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. The expression level of HOTAIR was normalized to that of GAPDH (forward: 5′-GGTGAAGGTCGGAGTCAACG-3′; reverse: 5′-CCATGTAGTTGAGGTCAATGAAG-3′) with the relative quantification 2−ΔΔCt formula. TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used for miR-148b-3p reverse transcription. Real-time PCR analysis was performed using TaqMan® Universal Master Mix II (Applied Biosystems, Foster City, CA, USA). The probes of miR-148b-3p (000471) and U6 (001973) were used. The expression level of miR-148b-3p was normalized to that of U6 with the relative quantification 2−ΔΔCt formula.
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