We purchased the cDNA microarray (MecDNA-HLivH087Su02 and MicDNA-HLivH087Su02) from Shanghai Outdo Biotech Co. The FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China) was utilized for the purification of total RNA. The PrimeScript™ RT reagent Kit (Takara, Japan) was used to convert 1 μg total RNA into cDNA through reverse transcription. For the examination of miRNA expression, we utilized the Hairpin-it™ miRNA Quantitation Kit (GenePharma, Suzhou, China) along with reverse transcription using specific stem-loop primers for miR-20a-5p, miR-195-5p, miR-17-5p, miR-106b-5p, and miR-93-5p. qRT-PCR was conducted using SYBR Green (Selleck, USA). The parameters for the two-stage amplification process were as follows: initial denaturation at a temperature of 95 °C for a duration of 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 30 s. GAPDH and U6 expression served as the internal reference for mRNA/lncRNA and miRNA, respectively. Supplementary Table 1 displays the specific primers for RNAs.
Hairpin it mirna quantitation kit
Manufactured by GenePharma
Sourced in China
The Hairpin-it™ miRNA Quantitation Kit is a laboratory equipment product designed for the quantitation of microRNA (miRNA) expression levels. The kit utilizes a Hairpin-it™ stem-loop RT-qPCR technology to enable sensitive and specific detection of mature miRNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (2 mentions)
Sybr green, by Selleck Chemicals (1 mentions)
Fastpure cell tissue total rna isolation kit v2, by Vazyme (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Rnaios plus, by Takara Bio (1 mentions)
2 protocols using hairpin it mirna quantitation kit
1
Profiling mRNA and miRNA Expression
2
Quantitative Analysis of miRNA Expression
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Total RNA was extracted using RNAios Plus (Takara, Otsu, Shiga, Japan). The quality and concentration of the isolated RNA were analyzed using a Nano drop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (800 ng) was converted to cDNA using the PrimeScript™ RT reagent Kit (Takara) with gDNA Eraser. To analyze the expression of miRNA, we performed reverse transcription with the miR-100-3p, miR-195-3p, miR-145-5p and miR-204-5p specific stem-loop primers and Hairpin-it™ miRNA Quantitation Kit (GenePharma, Suzhou, Jiangsu, China). We utilized a Light-Cycler 96 (Roche Diagnostic, Basel, Switzerland) to perform qRT-PCR using SYBR Green (Takara). The conditions of the two-step amplification reaction were as follows: preincubation at 95 °C for 30 s, then 40 cycles of 95 °C for 10 s and 60 °C for 30 s. GAPDH and U6 expression was utilized as the endogenous control for mRNA/lncRNA and miRNA, respectively. Expression level analysis was performed in accordance with the 2 -ΔΔC T method [19] . Specific primers for RNAs are shown in Supplementary Table 1.
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