We performed qRT-PCR for miR-200b expression with retinal samples. miRNA fractions were isolated from retinas using a miRNA isolation kit (CWbio, Co. Ltd.; cat. no. CW0627); U6-rRNA was used as an internal control. The primers sequences for miR-200b and U6-rRNA used for amplification are shown in Table 2 . PCR was carried out using a miRNA Real-time PCR Assay Kit (CWbio Co. Ltd; cat. no. CW214). After the reaction, the Ct values of the samples were calculated at the point at which they reached the threshold value during the process of PCR amplification, and miR-200b levels were quantified based on the ratio of miRNA/U6-rRNA using these 2-ΔΔ Ct method.
Mirna isolation kit
Manufactured by CWBIO
Sourced in China
The MiRNA isolation kit is a laboratory tool designed to extract and purify microRNA (miRNA) from biological samples. It functions to selectively isolate small RNA molecules, including miRNA, from various sample types such as cells, tissues, or body fluids. The kit utilizes column-based separation techniques to obtain high-quality miRNA for downstream applications, such as analysis and research.
Automatically generated - may contain errors
2 protocols using mirna isolation kit
1
Quantitative miR-200b Expression in Retina
2
Fruit Samples miRNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
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MiRNAs was extracted from fruit samples on the day move in and out of the cold storage (C01-03, LT01-03) and at OTP (COTP1-3, LTOTP1-3), with three biological replicates. Extraction was performed using an miRNA isolation kit (CWBIO, Beijing, China) following the instructions provided by the manufacturer. RNA quality was monitored by gel electrophoresis and an OD 260 /OD 280 ratio. High-quality RNA samples were used for miRNA library preparation and massive sequenced by an HiSeq 4000 SBS kit (300 cycles) (Illumina, San Diego, CA, USA).
Multiplexing was applied during sequencing. Libraries were constructed according to TruSeq TM Small RNA sample prep kit (Illumina, USA). Briefly, total RNA was purified by electrophoretic separation on a 15% TBE-urea denaturing polyacrylamide gel electrophoresis (PAGE) gel, and miRNA regions corresponding to the 18-32 nt bands in the marker lane were excised and recovered. Then, the18-32 nt miRNAs were ligated to a 5'-adaptor and a 3'-adaptor sequentially (TruSeq TM Small RNA sample prep kit). The adapter-ligated miRNAs were subsequently transcribed into cDNA and then PCR amplified for 12 cycles using the adaptor primers. The PCR products were purified for high-throughput sequencing.
Multiplexing was applied during sequencing. Libraries were constructed according to TruSeq TM Small RNA sample prep kit (Illumina, USA). Briefly, total RNA was purified by electrophoretic separation on a 15% TBE-urea denaturing polyacrylamide gel electrophoresis (PAGE) gel, and miRNA regions corresponding to the 18-32 nt bands in the marker lane were excised and recovered. Then, the18-32 nt miRNAs were ligated to a 5'-adaptor and a 3'-adaptor sequentially (TruSeq TM Small RNA sample prep kit). The adapter-ligated miRNAs were subsequently transcribed into cDNA and then PCR amplified for 12 cycles using the adaptor primers. The PCR products were purified for high-throughput sequencing.
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