Sybr green one step rt qpcr kit

Total RNA extraction from the lumbar nucleus pulposus specimens and cells was performed using an RNA extraction kit (Total RNA Purification kit; Sigma Aldrich; Merck KGaA). Total RNA was reverse transcribed into cDNA using the TaqMan^® MicroRNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RT reaction conditions were as follows: 37°C for 60 min, followed by 85°C for 5 min and 4°C. The cDNA samples were subjected to real-time PCR using a SYBR Green One-Step RT-qPCR kit (Thermo Fisher Scientific, Inc.) on an ABI 7900HT instrument (ABI; Thermo Fisher Scientific, Inc.). The qRCR reaction conditions were as follows: 95°C for 3 min, followed by 40 cycles at 95°C for 10 sec, 58°C for 30 sec and 72°C for 30 sec. The primer sequences for miR-222 were as follows: Forward primer: 5′-AGCUACAUCUGGCUACUGGGU-3′ and reverse primer: 5′-CCAGUAGCCAGAUGUAGCUUU-3′. The primer sequences for U6 were as follows: Forward primer: 5′-CTCGCTTCGGCAGCACAT-3′; reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′. All samples were tested in triplicate. The relative expression was measured using the 2^−ΔΔCq method (21 (link)). Gene expression results were normalized by the internal control U6.

The target mRNAs in cells and tissues were assessed by the RT-PCR method. The total RNA of cells and tissues was extracted using TRIzol® reagent (Thermo Fisher Scientific) according to the manufacturers’ instructions. β-actin was used as an internal control. qRT-PCR reactions were carried out using the SYBR Green One-Step RT-qPCR kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The primer sequences are detailed in Table S2.

The mRNA expression levels of SRPK1 in tissues or cells were assessed by the RT-qPCR method. The total RNA of tissues and cells was extracted using TRIzol^® reagent (Thermo Fisher Scientific) according to the manufacturers’ instructions. β-actin was used as an internal control. RT-qPCR reactions were carried out using SYBR Green One-Step RT-qPCR kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The relative mRNA expression levels were evaluated by the 2^−ΔΔCt method.20 (link) The primer sequences were as follows:
For SRPK1: forward, 5′-GGTGTGCCAGTCTTCCTCAAC-3′ reverse: 5′-GGTCCGTTATGTTCTTGCTCTTG-3′;
For β-actin: forward, 5′-GCGTGACATTAAGGAGAAG-3′ reverse: 5′-GAAGGAAGGCTGGAAGAG-3′.