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Hairpin it mirna qpcr kit

Manufactured by GenePharma
Sourced in China

The Hairpin-it™ miRNA qPCR kit is a laboratory equipment product designed for the quantitative detection of microRNA (miRNA) expression using real-time PCR (qPCR) technology. The kit provides a standardized and efficient method for miRNA quantification.

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2 protocols using hairpin it mirna qpcr kit

1

Gene Expression Analysis by qRT-PCR

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Total RNA was isolated using RNA iso Plus (Takara, D9108B). Real-time qRT-PCR was performed using reverse transcription kit (Takara, D6130) and SYBR Green PCR Master Mix (Toyobo, QPK-201). mRNA level were normalized to GAPDH. miRNA qPCR were performed according to manufacturer’s instructions using the Hairpin-it™ miRNA qPCR kit (Shanghai GenePharma Co. Ltd, QPM-091 for U6 snoRNA, QPM-040 for MIR155). Briefly, 4 µg total RNAs were reverse transcribed using mRNA specific RT primer, miRNA qPCR were performed using miRNA specific primer set to detect expression of mature MIR155. miRNA expression levels were normalized by Livak’s method and U6 snoRNA was used as endogenous control. Primers used in the present study were listed in Table S1.
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2

Quantitative Analysis of lncRNA and miRNA Expression

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Total RNA was extracted from tumor tissues and cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was generated with a reverse transcription kit from Takara (Dalian, China). Amplification reactions included 2 µl of Mix, 2 µl of RNA and 6 µl of ddH2O. The thermal cycling protocol was as follows: 37 °C for 15 min, 85 °C for 5 s and 4 °C for 15 min. The expression of SNHG16 and SLC2A4 was detected by using a SYBR Green Real-Time PCR Kit (Qiagen, Germany). miR-302b-3p expression was analyzed with a Hairpin-it miRNA qPCR kit (GenePharma, China). Amplification of the transcripts involved an initial denaturation step at 95 °C for 2 min followed by 40 cycles at 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The mRNA and miRNA expression data are presented as fold changes in the mRNA/miRNA abundance normalized to β-actin or U6 snRNA expression. The primer sequences used in this study are presented in Table 1.

Primer sequences used for qRT-PCR

GenePrimer sequences
 SNHG16
 F5ʹ-CAGAATGCCATGGTTTCCCC-3ʹ′
 R5ʹ-TGGCAAGAGACTTCCTGAGG-3ʹ
miR-302b-3p
 Loop5′-GTCGTATCCAGTCCAGGGACCGAGGACTGGATACGACCTACTA-3′
 F5′-GCGTAAGTGCTTCCATGTT-3′
 R5′-TCCAGGGACCGAGGA-3′
SLC2A4
 F5′-TGGCTGGGTTCTCCAACTG-3′
 R5′-CTGGAAACCCATGCCAATG-3′
β-actin
 F5′-ACCGAGCGCGGCTACAG-3′
 R5′-CTTAATGTCACGCACGATTTCC-3′
U6 snRNA
 F5′ -CTCGCTTCGGCAGCACA-3′
 R5′-AACGCTTCACGAATTTGCGT-3′
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