To verify sequencing results, 26 DEMs, including 24 known and two novel miRNAs, were selected and measured using SYBR green-based RT-qPCR. miRNA sequences and primers were synthesized by BGI Co. Ltd. (Shenzhen, China). Total RNA extracted from spleen samples was reverse transcribed into cDNA using a Mir-x™ miRNA First-Strand Synthesis and SYBR® RT-qPCR Kit (TaKaRa, Dalian, China) following the manufacturer’s instruction. Primers were used at 10 μM each to amplify target miRNAs in a 20 μl reaction mixture. RT-qPCR cycling conditions were as follows: 95 °C for 5 min; followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 15 s; and melting curve analysis from 60 °C to 95 °C. Three replicates were included for all reactions. Primers provided by the kit were used to amplify the snRNA U6 as a housekeeping miRNA to normalize miRNA expression. The expression of each miRNA relative to U6 was calculated using the 2−ΔΔCT method as described previously [71 (link)].
Mir x mirna first strand synthesis and sybr rt qpcr kit
Manufactured by Takara Bio
Sourced in China
The Mir-x™ miRNA First-Strand Synthesis and SYBR® RT-qPCR Kit is a laboratory equipment product designed for the detection and quantification of microRNA (miRNA) expression levels. The kit provides a complete solution for the reverse transcription and real-time PCR analysis of miRNA samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using mir x mirna first strand synthesis and sybr rt qpcr kit
1
Validating miRNA Sequencing with RT-qPCR
2
Validation of DEMs in Inflammation and Immunity
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To dissect the role of DEMs in in ammatory and immune, Cytoscape3.9.0 software was used to construct DEMs and immune and in ammatory-related genes regulatory networks at 10 and 15 DPI.
Quantitative real-time qPCR validation 9 DEMs, including 3 miRNAs in 10 DPI, 2 miRNAs in 15 DPI, and 4 miRNAs shared at 10 and 15 DPI, were selected and measured using SYBR green-based RT-qPCR to verify the sequencing results. MiRNA sequences in which uracil was replaced by thymine were used as the forward primers for the real-time PCR described in Table 1. The miRNA primers were synthesized by BGI Co. Ltd. (Shenzhen, China). The total RNA was extracted from cecal samples, and reverse transcribed into cDNA using a Mir-x™ miRNA First-Strand Synthesis and SYBR® RT-qPCR Kit (TaKaRa, Dalian, China) following the manufacturer's instruction. RT-qPCR cycling conditions were as follows: 95 °C for 5 mins; followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s; and melting curve analysis from 60 °C to 97 °C. All reactions were carried out with three repeats. U6 snRNA were used as internal reference gene for quantifying miRNA expression analysis. The expression of each miRNA relative to U6 was calculated using the 2-ΔΔCT method [17] .
Quantitative real-time qPCR validation 9 DEMs, including 3 miRNAs in 10 DPI, 2 miRNAs in 15 DPI, and 4 miRNAs shared at 10 and 15 DPI, were selected and measured using SYBR green-based RT-qPCR to verify the sequencing results. MiRNA sequences in which uracil was replaced by thymine were used as the forward primers for the real-time PCR described in Table 1. The miRNA primers were synthesized by BGI Co. Ltd. (Shenzhen, China). The total RNA was extracted from cecal samples, and reverse transcribed into cDNA using a Mir-x™ miRNA First-Strand Synthesis and SYBR® RT-qPCR Kit (TaKaRa, Dalian, China) following the manufacturer's instruction. RT-qPCR cycling conditions were as follows: 95 °C for 5 mins; followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s; and melting curve analysis from 60 °C to 97 °C. All reactions were carried out with three repeats. U6 snRNA were used as internal reference gene for quantifying miRNA expression analysis. The expression of each miRNA relative to U6 was calculated using the 2-ΔΔCT method [17] .
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