For the testing of the candidate miRNAs acquired on microarrays, RT-qPCR was performed. The total RNA was reverse transcribed using the miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech, Beijing, China) on a Proflex Base PCR System (Thermo Fisher Scientific, MA, USA). Real-time PCR reactions were performed on the PikoReal 96 (Thermo Scientific, MA, USA) in a 20-μL reaction volume containing 2 μL reverse transcription product, 10 μL 2×miRcute Plus miRNA Premix (with SYBR & ROX) (Tiangen Biotech), 0.4 μL PCR forward primer (5 μM, Tiangen Biotech), 0.4 μL reverse primer (10 μM, Tiangen Biotech), and 7.2 μL ddH2O. The reactions were incubated at 95°C for 15 min, followed by 40 cycles (94°C for 20 s and 60°C for 34 s). For the exogenous control, wascel-miR-39 was used; 1 μL of cel-miR-39 mimic (100 nM) was added into 200-μL plasma samples before RNA isolation. Each miRNA was calibrated against tocel-miR-39 to get a delta Ct (ΔCt) value for each miRNA (miRNA Ct value−cel-miR-39 Ct value). 2-ΔCt values in each group were used for the following statistical analysis. Expression fold changes were calculated busing the 2−ΔΔCt method.
Reverse primer
Manufactured by Tiangen Biotech
Sourced in United States, China
The reverse primer is a short, synthetic DNA sequence designed to complement the reverse, or complementary, strand of a DNA molecule. It serves as a starting point for DNA synthesis in the reverse direction during the polymerase chain reaction (PCR) process.
Automatically generated - may contain errors
Lab products found in correlation
Forward primer, by Tiangen Biotech (2 mentions)
Mircute plus mirna first strand cdna synthesis kit, by Tiangen Biotech (1 mentions)
Pikoreal96, by Thermo Fisher Scientific (1 mentions)
Proflex base pcr system, by Thermo Fisher Scientific (1 mentions)
Ddh2o, by Tiangen Biotech (1 mentions)
Bacterial dna kit, by Tiangen Biotech (1 mentions)
Taq dna polymerase, by Tiangen Biotech (1 mentions)
Dna marker 2000, by BioTeke (1 mentions)
3 protocols using reverse primer
1
miRNA Expression Analysis using RT-qPCR
2
Bacterial DNA Extraction and Phylogenetic Analysis
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DNA was extracted from each strain using the bacterial DNA kit according to the manufacturer's instructions (TIANGEN, Beijing, China). Primers are shown in Table 3 . The polymerase chain reaction (PCR) was performed in a 50 μL system including template DNA (2 μL), forward primer (2 μL), reverse primer (2 μL), 2× master mix (25 μL), and ddH2O (19 μL) (TIANGEN). PCR products were sequenced at the Huada Gene Company (Beijing, China). The sequencing results were compared using the Basic Local Alignment Search Tool (BLAST) program on NCBI. A phylogenetic tree was constructed using the neighbor-joining model as implemented in MEGA 5.0.
3
SSR-PCR Genotyping for Diversity Analysis
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SSR-polymerase chain reactions (PCRs) were carried out in 10-mL reaction volumes with 1X PCR buffer, 0.2 mM dNTPs, 1 U Taq DNA polymerase (Tiangen, Beijing, China), 0.5 mL forward primer (10 nM, Tiangen), 0.5 mL reverse primer (10 nm, Tiangen), and 0.5 mL DNA of each accession under the following PCR conditions: 5 min at 94°C, followed by 30 cycles for 30 s at 95°C, 30 s at the primer-specific annealing temperature, 30 s at 72°C, and final extension for 10 min at 72°C.
PCR products were separated on 8% polyacrylamide gels, and silver staining was conducted according to the method described by Zhang et al. (2000) . Molecular weights were estimated using a DNA marker (DNA Marker 2000, BioTeke Co., Beijing, China). SSR analysis was repeated at least twice. Clear bands from PCR products were recorded types 1-8. Faint bands were recorded as 0.
PCR products were separated on 8% polyacrylamide gels, and silver staining was conducted according to the method described by Zhang et al. (2000) . Molecular weights were estimated using a DNA marker (DNA Marker 2000, BioTeke Co., Beijing, China). SSR analysis was repeated at least twice. Clear bands from PCR products were recorded types 1-8. Faint bands were recorded as 0.
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