Liver miRNAs were extracted from the tissues using the MiRcute miRNA isolation kit (Tiangen Biotech Co., Ltd.), according to the manufacturer's protocol. Next, the concentration of RNA was quantified using a NanoDrop 1000 system (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). For RT into cDNA, the RNA was poly(A)-tailed and reverse transcribed into cDNA using random primers and the miRcute miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd.), following which qPCR was performed with the miRcute miRNA qPCR Detection kit (Tiangen Biotech Co., Ltd.) following the manufacturer's protocol with the forward primers presented in Table I . The universal reverse primers were synthesized and provided by Tiangen Biotech Co., Ltd. The PCR thermocycling conditions were as following: Denaturation at 95°C for 15 min, followed by 45 cycles of 94°C for 20 sec and 60°C for 34 sec. The cycle quantification (Cq) value was calculated using the second derivative maximum method (11 (link)). U6 small nuclear RNA was used as the internal control. The relative expression of each miRNA following normalization was determined as follows: Cq (U6)-Cq (miRNA).
Universal reverse primer
Manufactured by Tiangen Biotech
Sourced in China
The Universal Reverse Primer is a laboratory tool designed for use in reverse transcription-polymerase chain reaction (RT-PCR) experiments. It serves as a complementary oligonucleotide that binds to the 3' end of RNA molecules, enabling the synthesis of cDNA from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Mircute mirna isolation kit, by Tiangen Biotech (2 mentions)
Mircute mirna first strand cdna synthesis kit, by Tiangen Biotech (2 mentions)
Mircute mirna qpcr detection kit, by Tiangen Biotech (1 mentions)
Nanodrop 1000 system, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mircute mirna qpcr detection kit sybr green, by Tiangen Biotech (1 mentions)
Mircute microrna qpcr detection kit, by Tiangen Biotech (1 mentions)
Mircute microrna premix, by Tiangen Biotech (1 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Genespring software, by Agilent Technologies (1 mentions)
5 protocols using universal reverse primer
1
Quantitative Analysis of Liver miRNAs
2
Quantifying Colon Tissue miRNA Levels
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To determine miRNA levels, total RNA of colon tissue (cohort 3) was isolated with the miRcute miRNA Isolation Kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. miRNA was reverse transcribed into cDNA using a miRcute miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd.) at 37°C for 60 min. A miRcute miRNA qPCR Detection Kit (SYBR Green; Tiangen Biotech Co., Ltd.) was used for RT-qPCR analysis on an ABI 7500 fast real-time PCR system (Applied Biosystems) following the manufacturer's instructions. The cDNA (1 µl) was added to a 10-µl reaction system for amplification at 94°C for 2 min; followed by 42 cycles of 94°C for 20 sec and 60°C for 34 sec. All reactions were performed in triplicate. The specificity of the qPCR product was confirmed using melting curve analysis, and miRNAs with a Cq value >35 and a detection rate <75% in each group were excluded from further analysis. The relative expression of miRNA was normalized to that of the internal control U6. Relative expression was calculated using the 2−ΔΔCq method (15 (link)). The sequences of the forward primers are shown in Table II . Universal Reverse primers were obtained from Tiangen Biotech Co., Ltd.
3
Quantitative microRNA expression analysis
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The qPCR reaction was conducted with the miRcute microRNA qPCR Detection Kit (Tiangen, Beijing, China) on ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Each 20-μl qPCR reaction solution contained cDNA, 2× miRcute microRNA premix (with SYBR and ROX), the manufacturer-provided universal reverse primer, and a microRNA-specific forward primer (Tiangen, Beijing, China). The real-time PCR cycling conditions: 94°C for 2 min, 45 cycles at 94°C for 20 s, annealing at 60°C for 34 s, and extension at 72°C for 30 s. At the end of the real-time PCR reaction, a melting curve analysis was accomplished to ensure specific amplification of the expected PCR product.
The relative expression of the microRNAs was calculated from the equation log10 (2−ΔCt) with cel-miR-39. The ΔCT was calculated by subtracting the CT values of the cel-miR-39 from those of the microRNAs of interest [23 (link)].
The relative expression of the microRNAs was calculated from the equation log10 (2−ΔCt) with cel-miR-39. The ΔCT was calculated by subtracting the CT values of the cel-miR-39 from those of the microRNAs of interest [23 (link)].
4
Quantification of circRNA Expression
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Total RNA from PBMCs was reverse-transcribed into cDNA using a Prime-Script RT reagent kit with gDNA Eraser according to the manufacturer's protocol (Takara Bio, Inc.). RT-qPCR was performed using cDNA and SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.) using CFX96 Thermal Cycler equipment (Bio-Rad Laboratories, Inc.) following the manufacturer's instructions. The conditions were: 95°C for 30 sec, followed by 39 cycles of 95°C for 15 sec and 60°C for 30 sec. The primers used for quantitative real-time PCR were as follows: circPTPN22: F: 5′-AATTCTCACCAAATGTTCCCA-3′, R: 5′-AAGGTACATCATGGTCTGGC-3′; GAPDH: F: 5′-GGAGTCCACTGGCGTCTTC-3′, R: 5′-GCTGATGATCTTGAGGCTGTTG-3′. The levels of GAPDH were used to normalize the relative expression levels of circRNA.
The forward primers of microRNA and U6 used in the present study were synthesized by Tiangen Biotech Co., Ltd. and U6 small nuclear RNA was used as an endogenous control for miRNA normalization. The universal reverse primer was provided in the kit (Tiangen Biotech Co., Ltd.). Then, Ct values were obtained and calculated them by the 2−∆∆Cq method (30 (link)) for each sample. Each sample was repeated more than three times independently.
The forward primers of microRNA and U6 used in the present study were synthesized by Tiangen Biotech Co., Ltd. and U6 small nuclear RNA was used as an endogenous control for miRNA normalization. The universal reverse primer was provided in the kit (Tiangen Biotech Co., Ltd.). Then, Ct values were obtained and calculated them by the 2−∆∆Cq method (30 (link)) for each sample. Each sample was repeated more than three times independently.
5
Differential miRNA Expression in Exosomes
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MPDC-exosomes were used as a negative control. Through the miRNA microarray analysis, the differentially expressed miRNAs for KPC-exosomes were screened out. The microRNA microarray analysis was performed by OE Biotech. Co., Ltd. (Shanghai, China; http://www.oebiotech.com/ ). The Cy3-labeled RNA was purified before hybridization on an Agilent Mouse miRNA V21.0 chip (ID: 070155). Data analysis was initiated by first subtracting the background and then normalizing the signals using the GeneSpring software (version 13.1, Agilent). The threshold of significance was defined by the P value, with P ≤ 0.05 regarded as significant for the GO and KEGG analyses.
The validation of the microarray data was performed by RT-qPCR, using the StepOnePlus Real-Time PCR System (ABI, USA). The universal reverse primer was provided by TIANGEN BIOTECH CO. (Beijing, China). All other primer sequences are shown in Supplementary TableS1 . The differences in the expression of miRNAs between KPC-exosomes and MPDC-exosomes were analysed using the ΔΔCT method and normalized to U6 expression43 (link).
The validation of the microarray data was performed by RT-qPCR, using the StepOnePlus Real-Time PCR System (ABI, USA). The universal reverse primer was provided by TIANGEN BIOTECH CO. (Beijing, China). All other primer sequences are shown in Supplementary Table
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